Labelling and selection of molecules

ABSTRACT

A method of labelling molecules which includes providing in a common medium a label molecule, a marker ligand able to bind a member of a specific binding pair, such as an antigen, a sbp member, an enzyme able to catalyse binding of the label molecule to other molecules, the enzyme being associated with the marker ligand; causing or allowing binding of the marker ligand to the sbp member; and causing or allowing binding of the label molecule to other molecules in the vicinity of the marker ligand bound to the sbp member. The marker ligand may be an antibody or any specific binding molecule, such as a chemokine or cytokine. A complementary member of the specific binding pair may be included, e.g. an antibody, or a diverse population of such sbp members, e.g. antibodies, may be included within which those which bind the counterpart sbp member, e.g. antigen, may be labelled and subsequently isolated for manipulation and/or use. Suitable labels include biotin-tyramine with signal transfer being catalysed by hydrogen peroxidase. Cells, virus particles and other moieties may be labelled, for identification or obtention of proteins which interact or are in close proximity with a particular sbp member, or of cells of interest, or for enhancement of labelling, e.g. for cell sorting.

CROSS REFERENCE TO RELATED APPLICATION

This is a continuation of U.S. application Ser. No. 08/889,291, filedJul. 8, 1997 now U.S. Pat. No. 5,994,519.

The present invention relates to labelling and selection of molecules,such as members of a specific binding pair (sbp) able to bind acomplementary sbp member of interest, especially though not exclusivelya complementary sbp member for which an existing ligand is available. Inexemplary embodiments, the present invention relates to selection ofantibodies, or polypeptides comprising an antibody antigen bindingdomain, specific for an antigen of interest for which an existingbinding molecule, which may be an antibody, such as a monoclonalantibody, is already available. It involves deposition of a label orreporter molecule, such as biotin-tyramine, on molecules in the vicinityof a “marker ligand” which comprises for example a monoclonal antibody(specific for an antigen of interest) in association with an enzymewhich catalyzes such deposition. Molecules labelled in accordance withthe present invention may include binding members such as antibodieswhich bind the same binding target (e.g. antigen) as the marker ligandif such binding members are included in the reaction medium, the targetmolecule to which the marker ligand binds, which allows foridentification and/or purification of unknown antigen targets, and/orother molecules in the vicinity of the binding target aid/or the markerligand when bound to its binding target, e.g. on a cell surface on whichthe binding target is found, including molecules complexed with thebinding target, allowing for identification of novel protein-proteininteractions. There are also various advantages in labelling cells orother particles using the present invention, especially when the processis reiterated to augment the extent of labelling. Further aspects andembodiments of the invention are disclosed herein.

Numerous kinds of specific binding pairs are known, as epitomised by thepair consisting of antibody and antigen. Other specific binding pairsare discussed briefly infra and may equally be employed in the variousaspects of the present invention disclosed herein. For convenience,however, most of the discussion herein refers to antibody as the type of(first) specific binding pair (sbp) member whose selection is sought inperformance of methods of various embodiments of the invention,“antigen” as the complementary (second) sbp member of interest for whichspecific binding molecules may be sought to be selected and “markerligand” as the pre-existing binding molecule known to be able to bindthe complementary sbp member of interest Generally, the marker ligandcomprises an antibody antigen binding domain specific for thecomplementary sbp member of interest (e.g. antigen). Other suitablemarker ligands include hormones, cytokines, growth factors,neuropeptides chemokines, enzyme substrates and any other specificbinding molecule. Also present is a label or reporter molecule and anenzyme that catalyses binding of the label to other molecules in thevicinity.

Bearing this in mind, the present invention (in some embodiments) can besaid to have resulted from the inventors having identified a means toselect for antibodies binding to an antigen, e.g. on cell surfaces,other solid supports, or in solution, using a marker ligand for theantigen to guide the recovery of antibodies binding in proximity to themarker ligand. This provides means to label molecules which bind inclose proximity to a given defined ligand by transfer of a reportermolecule or label to the binding molecules. The defined ligand occupiesa specific epitope on the antigen and generally blocks that particularepitope, and epitopes overlapping it, from binding other antibodies.Thus; antibodies which are selected for are usually those which do notbind to the marker ligand epitope, but are those which bind neighbouringepitopes. Antibodies which bind the same epitope as the original markerligand may be obtained by an iterative process—using an antibodyobtained in one round of the process as a second marker ligand in afurther round or by using appropriate conditions, as discussed furtherbelow.

Signal transfer selection may be used to generate antibodies which bindto the same epitope as the marker ligand by re-iterating the selectionprocedure. Antibodies selected from the first round of signal transferselection may be used as new marker ligands for a subsequent round ofselection which is carried out in the absence of the original markerligand. This may be referred to as a “step-back” selection and may beused to select for antibodies which inhibit the original ligand binding.If the second stage of a step-back selection is carried out in thepresence of the original marker ligand antibodies which bind the markerligand-receptor complex, but not the receptor alone, may be selected.Such antibodies may be ligand agonists or antagonists. Of course, stepback selection need not be limited to Selection from antibody libraries;any pair of specific binding members can be used in such a procedure.

Antibodies which bind epitopes which are nearest to that bound by themarker ligand have the highest probability of becoming labelled, and theprobability of labelling decreases with distance from the marker ligandepitope. Advantageously, the present invention may expedite thepurification of such labelled molecules.

Transfer of the biotin tyramine reporter molecule may occur within up toabout 25 nm according to experimental results infra. The distance fromthe binding site of the original marker ligand may be increased byiteration of the signal transfer process, or by adapting the guidemolecule by the addition of a spacer between the guide molecule and theenzyme which catalyses the signal transfer. Such a spacer may be achemical linker, polymer, peptide, polypeptide, rigid bead, phagemolecule, or other particle.

Such a spacer may be of any suitable desired length, including about10-20 nm, about 20-40 nm, about 40-60 nm, about 60-100 nm, about 100 nmor more, such as about 500 nm or more up to about 1 μm or more.

Furthermore, the labelling and subsequent purification of bindingmolecules specific for antigen of interest which are displayed on thesurface of bacteriophage or other biological particles (see e.g.W092/01047) facilitates recovery of nucleic acid encoding the specificbinding molecules In so-called “phage display”, a binding molecule, e.g.antibody or antibody fragment, peptide or polypeptide, e.g. enzyme, isdisplayed on the surface of a virus particle which contains nucleic acidencoding the displayed molecule. Following selection of particles thatdisplay molecules with the desired binding specificity, the nucleic acidmay be recovered from the particles and used to express the specificbinding molecules or derivatives thereof, which may then be used asdesired.

Other display systems may be used instead of display on filamentousbacteriophage. Such systems include display on whole bacterial cells ormodified bacterial surface structures (Osuna et al. Crit. Rev.Microbiol., 1994, 20: 107-116; Lu et al., BioTechnology, 1995, 13:366-372) and eukaryotic viruses (Boublik et al. BioTechnology, 1995, 13:1079-1084; Sugiyama et al., FEBS Lett., 1995, L 359: 247-250).Bacteriophage display libraries may be generated using fusion proteinswith the gene III protein (e.g. Vaughan et al. Nature Biotechnology,.1996, 14: 309-314), or the major gene VIII coat protein (Clackson andWells, Trends Biotechnol., 1994, 12: 173-184), or the gene VI protein(Jespers et al, BioTechnology, 1995, 13: 378-382).

Herein it is shown that antibodies binding specifically to a giventarget antigen, e.g. expressed on the surface of cells, may be selectedfrom a large, diverse phage display library using an existing ligand ofthe desired antigen to guide the selection. It is also demonstrated thatthe desired antigen can be purified from the cells by chemicalmodification of the antigen in a reaction catalyzed by the existingligand. Antibodies to any antigen for which a known ligand exists may beobtained in this way, as may antibodies which bind specifically to theantigen-ligand complex rather than the antigen alone. In additionexisting ligands to unknown molecules (e.g. antigens) may be used asmarkers to guide selection of antibodies to the unknown molecule orpurification of the unknown molecule itself. Surface accessible regionsof an antigen may be identified by means of their accessibility tolabelling, e.g. biotinylation, Biotinylated molecules may be cleaved,e.g. proteolytically if they are peptidyl in nature, and biotinylatedfractions detected, e.g. following size fractionation. Furthermore, thelabelling of other molecules in the vicinity of the molecule to whichthe marker ligand binds allows for those other molecules to beidentified and/or purified for further study. It also allows forparticular moieties on which the binding target appears to be identifiedand/or purified, for instance one cell type displaying a particularantigen from among a complex mix of different cell types. Determinationof the extent of labelling which occurs in the vicinity of a themolecule to which the marker ligand binds may be used to determine thecopy number of that molecule, e.g. on a cell surface.

Selection of molecules in accordance with the present invention is notlimited to antigens on cell surfaces. For example, complex proteins withmultiple domains or subunits may be coated onto a solid support andligands specific for a particular domain or subunit may be used asmarker ligands to guide selection of antibodies to other neighbouringdomains or subunits. A domain or subunit may be conjugated, directly orindirectly, to the enzyme (e.g. HRP) and domain-domain orsubunit-subunit interactions used to guide selection. This may be termed“domain walking”. Marker ligands specific for particular epitopes on aprotein may also be used to guide the selection away from the markerligand epitope and to select for binding molecules which bind otherepitopes within the radius of labelling (e.g. about 25 nm forbiotinylation). This may be termed “epitope walking”, and example ofwhich is given in Example 8. A “step-back” selection may be carried out(as discussed elsewhere herein), generating a sbp member with the sameor overlapping epitope specificity as the original marker ligand.

Techniques of the present invention for selection of molecules, whichmay be known as “signal transfer selection”, need not be limited toantibody selection; selection from peptide libraries (e.g. displayed onphage) may be used to identify peptides with specific bindingcharacteristics for a given protein, which may be any binding domain ortype of ligand interaction, not just antibody/epitope. Example 14illustrates this using peptide libraries to epitope map an antibody(conjugated to HRP) in solution. Libraries or diverse populations ofproteins other than antibodies may be displayed on the surface of phageto allow isolation of novel proteins which bind to a protein inproximity to the marker ligand.

Signal transfer selection may also be used to chemically modify aparticular cell type possessing a specific antigen to facilitatepurification of that cell type from a background of other cells. Signaltransfer selection may also be applied to the humanisation of existingmonoclonal antibodies since Mab's which recognise an undefined antigenmay be used to target selection of human antibodies with a similarbinding capacity. This may involve the marker ligand including thebinding domain of a non-human antibody, such as a mouse monoclonalantibody, which may be conjugated directly or indirectly to an enzymesuch as HRP. Signal transfer selection may be used to obtain antibodiesfrom a human antibody library displayed on the surface of a suitablevirus, such as bacteriophage or retrovirus, or other biologicalparticle, which bind to the same antigen as the pre-existing non-humanantibody. Repeating the process (“step-back”) using an antibody obtainedin a first performance of the process as the marker ligand in a furtherperformance of the process may be used to obtain human antibodies whichbind to the same epitope as the original non-human antibody—a humanisedantibody. Ability of two binding molecules such as antibodies to bindthe same epitope may of course be assessed using an appropriatecompetition assay.

Signal transfer selection may be used to generate two antibodies, orother binding members, which bind adjacent epitopes on the same targetmolecule. This provides the potential to generate bispecific antibodies(such as “diabodies”) which may have higher affinities or otherdesirable biological properties (e.g. neutralising ability) which theindividual antibodies alone do not exhibit. Signal transfer selectionmay also be used with enzyme substrates to direct selection ofantibodies which bind enzyme active sites and which may be enzymeinhibitors or activators. Direct biotinylation of the enzyme active siteby the substrate may provide a tool to map amino acid residues importantin catalysis.

A local supply of hydrogen peroxide or other free radical precursor maybe generated by coupling the marker ligand to an enzyme which producesthe substrate for the free-radical generating enzyme, such as HRP, forexample, glucose oxidase or superoxide dismutase. This enables the localgeneration of radicalised biotin-tyramine or other label molecule in thevicinity of the free-radical generating enzyme. An active form offree-radical generating enzyme may be generated in response to a bindingevent, such as the bringing together of two subunits of the enzyme toproduce an active enzyme, or bringing together an activator of theenzyme with the enzyme itself. Radicalised label molecule such asbiotin-tyramine may be thus generated in response to binding events,which may be between specific cell types, proteins, or other specificbinding members.

TERMINOLOGY

Specific binding member

This describes a member of a pair of molecules which have bindingspecificity for one another. The members of specific binding pair may benaturally derived or synthetically produced. One member of the pair ofmolecules has an area on its surface, or a cavity, which specificallybinds to and is therefore complementary to a particular spatial andpolar organisation of the other member of the pair of molecules. Thusthe members of the pair have the property of binding specifically toeach other.

Examples of types of specific binding pairs are antigen-antibody,biotin-avidin/streptavidin, hormone-hormone receptor, receptor-ligand,enzyme-substrate.

Antibody

This describes an immunoglobulin whether natural or partly or whollysynthetically produced. The term also covers any polypeptide or proteinhaving a binding domain which is, or is homologous to, an antibodybinding domain. These can be derived from natural sources, or they maybe partly or wholly synthetically produced. Examples of antibodies arethe immunoglobulin isotypes and their isotypic subclasses; fragmentswhich comprise an antigen binding domain such as Fab, scfv, Fv, dAb, Fd;and diabodies.

It is possible to take monoclonal and other antibodies and usetechniques of recombinant DNA technology to produce other antibodies orchimeric molecules which retain the specificity of the originalantibody. Such techniques may involve introducing DNA encoding theimmunoglobulin variable region, or the complementarity determiningregions (CDRs), of an antibody to the constant regions, or constantregions plus framework regions, of a different immunoglobulin. See, forinstance, EP-A-184187, GB 2188638A or EP-A-239400. A hybridoma or othercell producing an antibody may be subject to genetic mutation or otherchanges, which may or may not alter the binding specificity ofantibodies produced.

As antibodies can be modified in a number of ways, the term “antibody”should be construed as covering any specific binding member or substancehaving a binding domain with the required specificity. Thus, this termcovers antibody fragments, derivatives, functional equivalents andhomologues of antibodies, including any polypeptide comprising animmunoglobulin binding domain, whether natural or wholly or partiallysynthetic. Chimeric molecules comprising an immunoglobulin bindingdomain, or equivalent, fused to another polypeptide are thereforeincluded. Cloning and expression of chimeric antibodies are described inEP-A-0120694 and EP-A-0125023.

It has been shown that fragments of a whole antibody can perform thefunction of binding antigens. Examples of binding fragments are (i) theFab fragment consisting of VL, VH, CL and CHl domains; (ii) the Fdfragment consisting of the VH and CHl domains; (iii) the Fv fragmentconsisting of the VL and VH domains of a single antibody; (iv) the dAhfragment (Ward, E. S. et al., Nature 341, 544-546 (1989)) which consistsof a VH domain; (v) isolated CDR regions; (vi) F(ab′)2 fragments, abivalent fragment comprising two linked Fab fragments (vii) single chainFv molecules (scFv), wherein a VH domain and a VL domain are linked by apeptide linker which allows the two domains to associate to form anantigen binding site (Bird et al, Science, 242, 423-426, 1988; Huston etal, PNAS USA, 85, 5879-5883, 1988); (viii) bispecific single chain Fvdimers (PCT/US92/09965=WO 93/11161) and (ix) “diabodies”, multivalent ormultispecific fragments constructed by gene fusion (WO94/13804; P.Holliger et al Proc. Natl. Acad. Sci. USA 90 6444-6448, 1993).

Diabodies are multimers of polypeptides, each polypeptide comprising afirst domain comprising a binding region of an immunoglobulin lightchain and a second domain comprising a binding region of animmunoglobulin heavy chain, the two domains being linked (e.g. by apeptide linker) but unable to associate with each other to form anantigen binding site: antigen binding sites are formed by theassociation of the first domain of one polypeptide within the multimerwith the second domain of another polypeptide within the multimer(WO94/13804).

Antigen binding domain

This describes the part of an antibody which comprises the area whichspecifically binds to and is complementary to part or all of an antigen.Where an antigen is large, an antibody may only bind to a particularpart of the antigen, which part is termed an epitope. An antibodyantigen binding domain may be provided by one or more antibody variabledomains. Preferably, an antigen binding domain comprises an antibodylight chain variable region (VL) and an antibody heavy chain variableregion (VH).

Specific

This refers to the situation in which one member of a specific bindingpair will not show any significant binding to molecules other than itsspecific binding partner (e.g. an affinity of about 1000×worse). Theterm is also applicable where eg an antigen binding domain is specificfor a particular epitope which is carried by a number of antigens, inwhich case the specific binding member carrying the antigen bindingdomain will be able to bind to the various antigens carrying theepitope.

Functionally equivalent variant form

This refers to a molecule (the variant) which although having structuraldifferences to another molecule (the parent) retains some significanthomology and also at least some of the biological function of the parentmolecule, e.g. the ability to bind a particular antigen or epitope.Variants may be in the form of fragments, derivatives or mutants. Avariant, derivative or mutant may be obtained by modification of theparent molecule by the addition, deletion, substitution or insertion ofone or more amino acids, or by the linkage of another molecule. Thesechanges may be made at the nucleotide or protein level. For example, theencoded polypeptide may be a Fab fragment which is then linked to an Fctail from another source. Alternatively, a marker such as an enzyme,flourescein, etc, may be linked.

Marker ligand

This refers to one member of a specific binding pair able to bindcomplementary sbp member. In embodiments of the present invention, it isused to guide catalysis of label or reporter molecule deposition at andaround its site of binding to the complementary other member of thespecific binding pair.

According to a first aspect of the present invention there is provided amethod of labelling molecules, the method including providing in acommon medium:

a label molecule;

a ligand (“first marker ligand”) able to bind a second member of aspecific binding pair (sbp);

a said second sbp member;

an enzyme able to catalyse binding of said label molecule to othermolecules, said enzyme being associated with said first marker ligand;

causing or allowing binding of said first marker ligand to said secondsbp member; and

causing or allowing binding of said label molecule to other molecules inthe vicinity of said first marker ligand bound to said second sbpmember.

A first member of a specific binding pair, such as an antibody, may beincluded, or a diverse population of such first sbp members includingone or more which bind the second sbp member. Molecules to which thelabel molecule binds may include a sbp member (“first sbp member”) whichbinds said second sbp member.

Molecules to which the label molecule binds may include a sbp member(“first sbp member”) which binds a molecule in the vicinity of saidsecond sbp member, as discussed further infra.

In preferred embodiments of the invention the first sbp member is apolypeptide comprising an antibody antigen binding domain, and thesecond, complementary sbp member is antigen. The marker ligand may be apolypeptide comprising an antibody antigen binding domain, such as amonoclonal antibody or cloned scFv, tab or other antibody fragment.

In a preferred embodiment of the present invention, the first member ofthe-specific binding pair is included and is labelled by binding of thelabel molecule. This allows identification and/or isolation of targetmolecules such as antibodies able to bind a substance of interest, suchas antigen. (The term “target molecules” may be used to refer tomolecules the identification of which is the object of the personskilled in the art operating the invention.) Such isolation may befacilitated if the label itself is a member of a specific binding pair.A preferred label exemplified herein is biotin, able specifically tobind avidin and streptavidin. Also exemplified is the use oflight-activatible streptavidin as the label.

Following binding of a sbp member label such as biotin to a target sbpmember (e.g. antibody), specific binding of the label to itscomplementary sbp member (e.g. streptavidin in the case of biotinlabelling) may be used in isolation of the target sbp member. Forinstance, streptavidin-coated magnetic beads may be added to the mediumor milieu, allowing streptavidin-biotin binding to take place, thenextracted using a magnet. Sbp members labelled with biotin may then berecovered from the beads.

Other suitable labels include photo-reactive compounds such asN-[N-4-azido-tetraflurobenzoyl)-biocytinyloxy]-succinimide, orphotoreactive crosslinking agents such as sulfor-SANPAH or SAND(sulfosuccinimidyl2-[m-azido-o-nitrobenzamido]-ethyl-1,3-dithiopropionate) in combinationwith streptavidin or biotin. Conveniently, biotin or other label isconjugated to tyramine, whose covalent binding to peptide molecules iscatalysed by oxygen free radicals generated by hydrogen peroxidase inthe presence of hydrogen peroxide. Instead of biotin-tyramine, labellingin performance of the present invention may employ other forms ofmodified tyramine including fluoresceinated tyramine or other freeradical reagents, such as p-hydroxyphenylpropionyl-biocytin andbiotynil-coumarin galactose. Labels such as biotin (e.g. asbiotin-tyramine) may be preferred over photo-reactive labels, e.g.because of ease of handling, though Example 10 below demonstratesoperation of the present invention using a label whose binding islight-activated, i.e. SAND linked to streptavidin. An advantage of usinga light-activatable label, such as streptavidin-SAND, is the distanceover which this label can be deposited. The linker between thestreptavidin and SAND is 1.8 nm so the proximity within which thestreptavidin is deposited is up to a maximum of about 1.8 nm, comparedwith a radius of up to about 25 nm of biotinylation which is obtainablewith biotin-tyramine.

The enzyme that catalyses binding of the label molecule to othermolecules may be associated with the marker ligand by any suitable meansavailable in the art. It may be conjugated directly, e.g. via a peptidebond (in which case a fusion protein comprising marker ligand and enzymemay be produced by expression from encoding nucleic acid), or bychemical conjugation of the marker ligand and enzyme, or indirectly.Indirect conjugation of enzyme and marker ligand may conveniently beachieved using a further binding molecule that forms a specific bindingpair with the marker ligand. For example, the marker ligand may be amouse monoclonal antibody, or may comprise a mouse antibody sequence,and the enzyme may be provided conjugated to an anti-mouse antibody orantibody antigen binding domain (e.g. as a fusion protein). Binding ofanti-mouse antibody to the mouse monoclonal, itself binding the antigenof interest (second sbp member), brings the conjugated enzyme into closeproximity with the antigen and any molecules in the medium or milieuable to bind the antigen (e.g. target antibodies), allowing the enzymeto catalyse labelling of such molecules (e.g. target antibodies) and/orthe antigen. Labelled molecules may be identified and/or isolated forinvestigation and/or use.

As mentioned already, the first sbp member when provided in the reactionmilieu may be one of a diverse population of that type of sbp memberwith different binding specificities. Such a population may be providedby expression from a genetically diverse repertoire of nucleic acidsequences. In the case of antibody antigen binding domains, these may beprovided by exression from a repertoire of rearranged or unrearrangedimmunoglobulin sequences from an organism (preferably human) which hasor has not been immunised with the antigen of interest. A repertoire ofsequences encoding antibody antigen binding domains (VH and/or VL) mayadditionally or alternatively be provided by any of artificialrearrangement of V, J and D gene segments, mutation in vitro or in vivo,in vitro polynucleotide synthesis and/or any other suitable techniqueavailable in the art. Suggested references include Vaughan et al.,(1996) Nature Biotechnology 14: 309-314; Griffiths et al., (1993) EMBOJ. 12: 725-734.

Conveniently, a diverse population of binding molecules is provideddisplayed on the surface of a biological particle such as a virus, e.g.bacteriophage, each particle containing nucleic acid encoding thebinding molecule displayed on its surface WO92/01041 discloses in detailvarious formats for “phage display” of polypeptides and peptide bindingmolecules, such as antibody molecules, including scfv, Fab and Fvfragments, and enzymes, both monomeric and polymeric. Followinglabelling of phage displaying a target sbp member able to bindcomplementary sbp member of interest, and isolation of these from thereaction medium or milieu as discussed, nucleic acid may be recoveredfrom phage particles. This nucleic acid may be sequenced if desired.

Other display systems, e.g. on bacterial cells or retroviruses, areapplicable, as has been mentioned already.

The nucleic acid taken from the particle, or its nucleotide sequence,may be used to provide nucleic acid for production of the encodedpolypeptide or a fragment or derivative thereof in a suitable expressionsystem, such as a recombinant host organism. A derivative may differfrom the starting polypeptide from which it is derived by the addition,deletion, substitution or insertion of amino acids, or by the linkage ofother molecules to the encoded polypeptide. These changes may be made atthe nucleotide or protein level. For example the encoded polypeptide maybe a Fab fragment which is then linked to an Fc tail from anothersource. Alternatively markers such as enzymes, flouresceins etc may belinked to eg Fab, scFv fragments.

Systems for cloning and expression of a polypeptide in a variety ofdifferent host cells are well known. Suitable host cells includebacteria, mammalian cells, yeast and baculovirus systems. Mammalian celllines available in the art for expression of a heterologous polypeptideinclude Chinese hamster ovary cells, HeLa cells, baby hamster kidneycells and many others. A common, preferred bacterial host is E. coli.

Suitable vectors can be chosen or constructed, containing appropriateregulatory sequences, including promoter sequences, terminatorfragments, polyadenylation sequences, enhancer sequences, marker genesand other sequences as appropriate. Vectors may be plasmids, viral e.g.‘phage, or phagemid, as appropriate. For further details see, forexample, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrooket al., 1989, Cold spring Harbor Laboratory Press. Many known techniquesand protocols for manipulation of nucleic acid, for example inpreparation of nucleic acid constructs, mutagenesis, sequencing,introduction of DNA into cells and gene expression, and analysis ofproteins, are described in detail in Short Protocols in MolecularBiology, Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992.The disclosures of Sambrook et al. and Ausubel et al. are incorporatedherein by reference.

The expression end product may be used to prepare a compositioncomprising the expression end product or a derivative thereof andoptionally one or more further components such as a pharmaceuticallyacceptable vehicle, carrier or excipient, which may for example be usedas a therapeutic or prophylactic medicament or a diagnostic product.

In some embodiments of the present invention, the second sbp member (towhich the marker ligand binds—e.g. antigen) is labelled. This is usefulif the target molecule is an unknown antigen/receptor for the knownmarker ligand (e.g. monoclonal antibody or the natuxal ligand for theantigen/receptor). In such case, the first sbp member may be omittedfrom the reaction medium or milieu. Following labelling of the secondsbp member it may be identified and/or isolated in accordance withprocedures disclosed herein.

According to a further aspect of the present invention there is providedreaction medium or milieu containing:

a member of said specific binding pair;

a label molecule;

a ligand (“marker ligand”) able to bind said sbp member;

an enzyme able to catalyse binding of said label molecule to othermolecules, said enzyme being associated with said marker ligand;

as provided in methods according to the invention. A further sbp member(designated “first”) may be present, in which case the marker ligand isable to bind complementary “second” sbp member.

A further aspect of the present invention provides a sbp memberidentified as having ability to bind complementary sbp member ofinterest and/or isolated using a method as disclosed herein, including areceptor or ligand identified and/or isolated as disclosed, andcompositions comprising such an identified and/or isolated sbp memberand nucleic acid encoding the identified and/or isolated sbp member.

The present invention generally provides for any specific binding memberidentified by virtue of its ability to bind to complementary sbp memberin close proximity (e.g. less than about 25 nm, and possibly less thanabout 20 nm, less than about 15 nm, less than about 10 nm, about 5-10 nmor about 5 nm) to an existing defined ligand, which may be termed a“marker ligand” and is used to guide catalysis of reporter moleculedeposition on to the specific binding member.

The invention also provides for the use of the methods and meansprovided herein for the selection of phage-displayed sbp members, e.g.antibodies, peptides or proteins, also the selection or identificationof unknown receptors using a known ligand, either by directed labellingof the receptor, or by production of an antibody against the receptor,followed by immuno-purification.

The invention also provides for the use of signal transfer selection inan iterative manner, i.e. using one or more sbp members selected in acycle to select for further sbp members. This may be used to select sbpmembers which are capable of acting as antagonists or agonists to theoriginal marker ligand used in the first stage of the selection.

Cell-surface or other receptors may be identified in a process accordingto the present invention by conjugating a ligand for the uncharacterisedreceptor (e.g. the natural of the receptor) with an enzyme able tocatalyse binding of the label molecule. Binding of the ligand to thereceptor, e.g. on cells expressing it, may then be carried out in thepresence or absence of sbp members, such as antibodies, particularly alibrary of sbp members, e.g. displayed on phage, and the label molecule.The natural ligand may transfer the signal molecule directly onto theunknown receptor. Labelled receptor may then be directly purified, e.g.from a cell extract, and may be protein sequenced. In the presence ofthe sbp members, e.g. a library of antibodies displayed on phage, signaltransfer will generate labelled sbp members which are able to bind thereceptor. These may then be used to generate purified receptor byaffinity purification.

The invention also provides for the use of such processes to identifyunknown ligands for known receptors, either by directed labelling of theligand, or by production of an antibody directed against the ligandfollowed by immuno-purification.

Further provided by the invention is the use of signal transferselection to guide the selection of antibodies to a given epitope,domain or subunit of a protein or complex by an existing ligand orantibody which recognises a neighbouring epitope, domain or subunit.Existing sbp's (e.g. monoclonal antibodies) to a defined but perhapsundesirable epitope, subunit or region of a protein complex may beconjugated to an enzyme capable of catalysing binding of the labelmolecule to other molecules. These conjugated sbp's may then be used todirect signal transfer of the label to other sbp members, e.g.antibodies (e.g. on phage), binding to the same antigen but atnon-identical, non-overlapping, but neighbouring epitopes which may beon adjacent subunits of a protein, or on adjacent regions of a proteincomplex.

Signal transfer selection may be used to obtain antibodies or otherbinding molecules which bind to the same epitope as the marker ligand.For example, sub-saturating amounts of the marker ligand may be added toa mutlimeric protein and the marker ligand may then direct selection ofbinding specificities recognising the same epitope as the marker ligand,but on a neighbouring subunit, or copy of the multimer. The markerligand may be capable of labelling binding species which bind to thesame epitope if labelling occurs concomitantly with the marker ligandbeing competed off the target protein by the species which is beingselected for.

Another application of the process is that of selecting for antibodiesor other ligands which bind to a particular cell structure or cell type.

Further aspects of the present invention arise from the gene cloningwork described in Example 16. Encoding nucleic acid, isolatedpolypeptides, specific binding molecules for the polypeptide and othermolecules which interact with the polypeptide, particularly those whichmodulate its function, e.g. interfere with its association with CC-CKR5and/or other polypeptide in the vicinity of CC-CKR5 on the surface ofCD4+ cells, other molecules which interact with the polypeptide, andmethods and uses of these are all provided by the present invention.

Nucleic acid according to this aspect of the present invention mayinclude or consist essentially of a nucleotide sequence encoding apolypeptide which includes an amino acid sequence shown in FIG. 8.

The coding sequence may be that shown in FIG. 8, or it may be a mutant,variant, derivative or allele of the sequence shown. The sequence maydiffer from that shown by a change which is one or more of addition,insertion, deletion and/or substitution of one or more nucleotides ofthe sequence shown. Changes to a nucleotide sequence may result in anamino acid change at the protein level, or not, as determined by thegenetic code.

Thus, nucleic acid according to the present invention may include asequence different from the sequence shown in FIG. 8 yet encode apolypeptide with the same amino acid sequence. The polypeptide mayinclude a sequence of about 60 contiguous amino acids from FIG. 8, morepreferably about 70 contiguous amino acids, more preferably about 80. Anamino acid sequence from the second reading frame may be preferred. Astop codon occurs in this frame at nucleotide 251, so in a preferredembodiment the polypeptide includes a contiguous sequence of amino acidsencoded by the nucleotide sequence of the second reading frame of FIG. 8up to said stop codon. Usually, additional amino acids are includedN-terminal to the amino acid sequence shown.

On the other hand, the encoded-polypeptide may include an amino acidsequence which differs by one or more amino acid residues from therelevant amino acid sequence shown in FIG. 8. Nucleic acid encoding apolypeptide which is an amino acid sequence mutant, variant, derivativeor allele of a sequence shown in FIG. 8 is further provided by thepresent invention.

Nucleic acid encoding such a polypeptide may show at the nucleotidesequence and/or encoded amino acid level greater than about 50% homologywith the relevant coding/amino acid sequence shown in FIG. 8, greaterthan about 60% homology, greater than about 70% homology, greater thanabout 80% homology, greater than about 90% homology or greater thanabout 95% homology.

As is well-understood, homology at the amino acid level is generally interms of amino acid similarity or identity. Similarity allows for“conservative variation”, such as substitution of one hydrophobicresidue such as isoleucine, valine, leucine or methionine for another,or the substitution of one polar residue for another, such as argininefor lysine, glutamic for aspartic acid, or glutamine for asparagine.Similarity may be as defined and determined by the TBLASTN program, ofAltschul et al. (1990) J. Mol. Biol. 215: 403-10, which is in standarduse in the art. Homology may be over the full-length of the relevantamino acid sequence of FIG. 8, or may more preferably be over acontiguous sequence of about 20, 25, 30, 40, 50, 60, 70, 80 or moreamino acids, compared with the relevant amino acid sequence of FIG. 8.

At the nucleic acid level, homology may be over the full-length or morepreferably by comparison with the a contiguous nucleotide codingsequence within the sequence of FIG. 8 of about 50, 60, 70, 80, 90, 100,120, 150, 180, 210, 240 or more nucleotides.

Generally, nucleic acid according to the present invention is providedas an isolate, in isolated and/or purified form, or free orsubstantially free of material with which it is naturally associated,such as free or substantially free of nucleic acid flanking the gene inthe human genome, except possibly one or more regulatory sequence(s) forexpression. Nucleic acid may be wholly or partially synthetic and mayinclude genomic DNA, cDNA or RNA. Where nucleic acid according to theinvention includes RNA, reference to the sequence shown should beconstrued as reference to the RNA equivalent, with U substituted for T.

Nucleic acid sequences encoding all or part of the gene and/or itsregulatory elements can be readily prepared by the skilled person usingthe information and references contained herein and techniques known inthe art (for example, see Sambrook, Fritsch and Maniatis, “MolecularCloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989,and Ausubel et al, Short Protocols in Molecular Biology, John Wiley andSons, 1992).

The sequence information provided in FIG. 8 enables cloning of thefull-length human coding sequence. The present invention provides amethod of obtaining nucleic acid of interest, the method includinghybridisation of a probe having the sequence shown in FIG. 8 or acomplementary sequence, or a suitable fragment of either, to targetnucleic acid. Hybridisation is generally followed by identification ofsuccessful hybridization and isolation of nucleic acid which hashybridised to the probe, which may involve one or more steps of PCR. Thenucleic acid sequences provided herein readily allow the skilled personto design PCR primers for amplification of the full-length sequence.

Nucleic acid according to the present invention is obtainable using oneor more oligonucleotide probes or primers designed to hybridise with oneor more fragments of the nucleic acid sequence shown in FIG. 8particularly fragments of relatively rare sequence, based on codon usageor statistical analysis. A primer designed to hybridise with a fragmentof the nucleic acid sequence shown in FIG. 8 may be used in conjunctionwith one or more oligonucleotides designed to hybridise to a sequence ina cloning vector within which target nucleic acid has been cloned, or inso-called “RACE” (rapid amplification of cDNA ends) in which cDNA's in alibrary are ligated to an oligonucleotide linker and PCR is performedusing a primer which hybridises with the sequence shown in FIG. 8 and aprimer which hybridises to the oligonucleotide linker.

Such oligonucleotide probes or primers, as well as the full-lengthsequence. (and mutants, alleles, variants and derivatives) are alsouseful in screening a test sample containing nucleic acid for thepresence of alleles, mutants and variants, with diagnostic and/orprognostic implications.

Nucleic acid isolated and/or purified from one or more cells (e.g.human) or a nucleic acid library derived from nucleic acid isolatedand/or purified from cells (e.g. a cDNA library derived from mRNAisolated from the cells), may be probed under conditions for selectivehybridisation and/or subjected to a specific nucleic acid amplificationreaction such as the polymerase chain reaction (PCR), as discussed.

In the context of cloning, it may be necessary for one or more genefragments to be ligated to generate a full-length coding sequence. Also,where a full-length encoding nucleic acid molecule has not beenobtained, a smaller molecule representing part of the full molecule, maybe used to obtain full-length clones. Inserts may be prepared frompartial cDNA clones and used to screen cDNA libraries. The full-lengthclones isolated may be subcloned into expression vectors and activityassayed by transfection into suitable host cells, e.g. with a reporterplasmid.

Those skilled in the art are well able to employ suitable conditions ofthe desired stringency for selective hybridisation, taking into accountfactors such as oligonucleotide length and base composition, temperatureand so on.

On the basis of amino acid sequence information, oligonucleotide probesor primers may be designed, taking into account the degeneracy of thegenetic code, and, where appropriate, codon usage of the organism fromthe candidate nucleic acid is derived. An oligonucleotide for use innucleic acid amplification may have about 10 or fewer codons (e.g. 6, 7or 8), i.e. be about 30 or fewer nucleotides in length (eg. 18, 21 or24). Generally specific primers are upwards of 14 nucleotides in length,but not more than 18-20. Those skilled in the art are well versed in thedesign of primers for use processes such as PCR. Various techniques forsynthesizing oligonucleotide primers are well known in the art,including phosphotriester and phosphodiester synthesis methods.

A further aspect of the present invention provides an oligonucleotide orpolynucleotide fragment of the nucleotide sequence shown in FIG. 8, or acomplementary sequence, in particular for use in a method of obtainingand/or screening nucleic acid. Some preferred oligonucleotides have asequence shown in FIG. 8 or a sequence which differs from any of thesequences shown by addition, substitution, insertion or deletion of oneor more nucleotides, but preferably without abolition of ability tohybridise selectively with nucleic acid with the sequence shown in FIG.8, that is wherein the degree of homology of the oligonucleotide orpolynucleotide with one of the sequences given is sufficiently high.

Nucleic acid according to the present invention may be used in methodsof gene therapy, for instance in treatment of individuals with the aimof preventing or curing (wholly or partially) a disease. This may easeone or more symptoms of the disease.

A convenient way of producing a polypeptide according to the presentinvention is to express nucleic acid encoding it, by use of the nucleicacid in an expression system.

Accordingly, the present invention also encompasses a method of making apolypeptide (as disclosed), the method including expression from nucleicacid encoding the polypeptide (generally nucleic acid according to theinvention). This may conveniently be achieved by growing a host cell inculture, containing such a vector, under appropriate conditions whichcause or allow expression of the polypeptide. Polypeptides may also beexpressed in in vitro systems, such as reticulocyte lysate.

Systems for cloning and expression of a polypeptide in a variety ofdifferent host cells are well known. Suitable host cells includebacteria, eukaryotic cells such as mammalian and yeast, and baculovirussystems. Mammalian cell lines available in the art for expression of aheterologous polypeptide include Chinese hamster ovary cells, HeLacells, baby hamster kidney cells, COS cells and many others. A common,preferred bacterial host is E. coli.

Nucleic acid may be introduced into a host cell and this may be followedby causing or allowing expression from the nucleic acid, e.g. byculturing host cells (which may include cells actually transformedalthough more likely the cells will be descendants of the transformedcells) under conditions for expression of the gene, so that the encodedpolypeptide is produced. If the polypeptiae is expressed coupled to anappropriate signal leader peptide it may be secreted from the cell intothe culture medium. Following production by expression, a polypeptidemay be isolated and/or purified from the host cell and/or culturemedium, as the case may be, and subsequently used as desired, e.g. inthe formulation of a composition which may include one or moreadditional components, such as a pharmaceutical composition whichincludes one or more pharmaceutically acceptable excipients, vehicles orcarriers (e.g. see below).

The skilled person can use the techniques described herein and otherswell known in the art (for which see e.g. the Sambrook and Ausubelreferences cited herein) to produce large amounts of polypeptide, orfragments or active portions thereof, for use as pharmaceuticals, in thedevelopments of drugs and for further study into its properties and rolein vivo.

Thus, a further aspect of the present invention provides a polypeptidewhich includes an amino acid sequence shown in FIG. 8 as discussed,which may be in isolated and/or purified form, free or substantiallyfree of material with which it is naturally associated, such as otherpolypeptides or such as human polypeptides other than polypeptide or(for example if produced by expression in a prokaryotic cell) lacking innative glycosylation, e.g. unglycosylated.

Polypeptides which are amino acid sequence variants, alleles,derivatives or mutants are also provided by the present invention, ashas been discussed. Preferred such polypeptides have function, that isto say have one or more of the following properties: immunologicalcross-reactivity with an antibody reactive with a polypeptide for whichthe sequence is given in FIG. 8; sharing an epitope with a polypeptidefor which the amino acid sequence is shown in FIG. 8 (as determined forexample by immunological cross-reactivity between the two polypeptides.

The present invention also includes active portions fragments,derivatives and functional mimetics of the polypeptides of theinvention. A fragment of the polypeptide may be a stretch of amino acidresidues of at least about five to seven contiguous amino acids, oftenat least about seven to nine contiguous amino acids, typically at leastabout nine to 13 contiguous amino acids and, most preferably, at leastabout 20 to 30 or more contiguous amino acids. Fragments of thepolypeptide sequence antigenic determinants or epitopes useful forraising antibodies to a portion of the amino acid sequence.

A polypeptide, peptide fragment, allele, mutant or variant according tothe present invention may be used in phage display or other technique(e.g. involving immunisation) in obtaining specific antibodies.Antibodies are useful in purification and other manipulation ofpolypeptides and peptides, diagnostic screening and therapeuticcontexts.

The provision of the novel polypeptides enables for the first time theproduction of antibodies able to bind it specifically, and by proceduresother than the signal transfer selection which led to its identificationand the isolation of antibody CD4E1 as described in Example 16.Accordingly, a further aspect of the present invention provides anantibody able to bind specifically to a polypeptide including a sequencegiven in FIG. 8.

Such antibodies may be obtained by selection on peptides or proteinsincluding amino acid sequences of FIG. 8, e.g. using phage displaylibraries as in WO92/01047, or by using such peptides or proteins toimmunise animals and obtain monoclonal antibodies or polyclonalantisera.

Antibodies identified, e.g. by phage display, may then be used toidentify further proteins, e.g. receptor molecules, which may becomplexed with the protein including the amino acid sequence of FIG. 8,using techniques of signal transfer selection as disclosed herein.

cDNA expression libraries, for example displayed on phage, may be usedin conjunction with signal transfer selection to identify ligands whichbind molecules, such as receptors, in the vicinity of protein includingthe amino acid sequence of FIG. 8. An antibody, e.g. with a myc tag, maybind to the protein on the surface of CD4 lymphocytes. thephage-displayed cDNA expression library may be added, followed by theantibody 9E10 (which binds to the myc tag) conjugated to HRP. Additionof biotin-tyramine would then lead to the labelling of molecules in thevicinity of the antibody, including phage expressing receptor ligands.The antibody CD4E1 would be suitable for this.

The polypeptides, antibodies, peptides and nucleic acid of the inventionmay be formulated in a composition. Such a composition may include, inaddition to one of the above substances, a pharmaceutically acceptableexcipient, carrier, buffer, stabiliser or other materials well known tothose skilled in the art. Such materials should be non-toxic and shouldnot interfere with the efficacy of the active ingredient. The precisenature of the carrier or other material may depend on the route ofadministration, e.g. oral, intravenous, cutaneous or subcutaneous,nasal, intramuscular, intraperitoneal routes.

A polypeptide according to the present invention may be used inscreening for molecules which affect or modulate its activity orfunction, including ability to interact or associate with anothermolecule, such as CC-CKR5 or other molecule, e.g. on the surface of CD4+cells. Such molecules may be useful in a therapeutic possibly includingprophylactic) context.

A method of screening for a substance which modulates activity of apolypeptide may include contacting one or more test substances with thepolypeptide in a suitable reaction medium, testing the activity of thetreated polypeptide and comparing that activity with the activity of thepolypeptide in comparable reaction medium untreated with the testsubstance or substances. A difference in activity between the treatedand untreated polypeptides is indicative of a modulating effect of therelevant test substance or substances.

Combinatorial library technology provides an efficient way of testing apotentially vast number of different substances for ability to modulateactivity of a polypeptide. Such libraries and their use are known in theart. The use of peptide or protein libraries may be preferred.

As an alternative to using signal transfer selection to identifymolecules which interact with protein including an amino acid sequenceshown in FIG. 8, test substances may be screened for ability to interactwith the polypeptide, e.g. in a two-hybrid system (which requires thatboth the polypeptide and the test substance can be expressed, e.g. in acell such as a yeast or mammalian cell, from encoding nucleic acid).This may be used as a coarse screen prior to testing a substance foractual ability to modulate activity of the polypeptide. The screen maybe used to screen test substances for binding to a specific bindingpartner, to find mimetics of polypeptide, e.g. for testing as anti-tumortherapeutics. Two-hybrid screens may be used to identify a substanceable to modulate, e.g. interfere with, interaction between twopolypeptides or peptides.

The two-hybrid screen assay format is described by Fields and Song,1989, Nature 340; 245-246. This type of assay format can be used in bothmammalian cells and in yeast. Various combinations of DNA binding domainand transcriptional activation domain are available in the art, such asthe LexA DNA binding domain and the VP60 transcriptional activationdomain, and the GAL4 DNA binding domain and the GAL4 transcriptionalactivation domain. Suitable fusion constructs are produced forexpression within the assay system. When screening for a susbstance ableto modulate an interaction between two components, test substances (e.g.in a combinatorial peptide library) may be expressed from a thirdconstruct.

Following identification of a substance which modulates or affectspolypeptide activity and/or its ability to interact with or associatewith another molecule, the substance may be investigated further.Furthermore, it may be manufactured and/or used in preparation, i.e.manufacture or formulation, of a composition such as a medicament,pharmaceutical composition or drug. These may be administered toindividuals.

Thus, the present invention extends in various aspects not only to asubstance identified using a nucleic acid molecule as a modulator ofpolypeptide activity, in accordance with what is disclosed herein, butalso a pharmaceutical composition, medicament, drug or other compositioncomprising such a substance, a method comprising administration of sucha composition to a patient, e.g. for treatment (which may includepreventative treatment) of cancer, use of such a substance inmanufacture of a composition for administration, e.g. for treatment ofcancer, and a method of making a pharmaceutical composition comprisingadmixing such a substance with a pharmaceutically acceptable excipient,vehicle or carrier, and optionally other ingredients.

Further aspects of the invention and embodiments will be apparent tothose skilled in the art. All documents mentioned herein areincorporated by reference. In order that the present invention may befully understood the following examples are provided by way ofexemplification only and not by way of limitation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic representation of a process according to oneembodiment of the present invention. A target antibody able to bind theantigen of interest (CEA) is labelled by biotinylation because it bindsthe antigen in the region of binding of a marker ligand which comprisesa monoclonal antibody specific for CEA joined to the enzyme hydrogenperoxidase. In the presence of hydrogen peroxide, the hydrogenperoxidase catalyses binding of biotin-tyramine to molecules in thevicinity of the enzyme, including the target antibody.(CEA—carcinoembronic antigen; HRP—horseradish peroxidase, A hydrogenperoxidase; BT—Biotin-tyramine.)

FIG. 2 illustrates results obtained in experiments described in Example12, showing the distribution of gold particles at the ends of page. Fordifferent numbers of beads per phage end the frequency is plotted. Theaverage number of particles per phage was 6.6, the detected range 5 nmto 25 nm. The diameter of a globular protein is 4 nm.

FIGS. 3(a) and (b) illustrate a “step-back” selection scheme asexemplified experimentally in Example 13.

FIG. 3(a) illustrates a process in which HRP-marker ligand conjugatedirects the signal transfer of biotin tyramine (BT) onto phage bindingaround the ligand. Biotinylated phage are then allowed to bind cells inthe absence of ligand, as shown in FIG. 3(b).

FIG. 3(b) shows binding of biotinylated phage in the absence of theoriginal marker ligand. Streptavidin-HRP is added and a new aliquot ofphage library then added (illustrated in black) which can then bebiotinylated by signal transfer and selected. In the illustratedembodiment, the selected phage mimics the ligand and inhibits itsbinding to cells.

FIG. 4 shows results of flow cytometry experiments described in Example18. The peak position (i.e. a measure of the fluorscence achieved)obtained using different biotin tyramine concentrations (in μg/ml) isplotted.

FIG. 5 shows the fluorescence shifts resulting from two flow cytometryreadings, one for a sample subject to one biotin tyramine treatment, theother for a sample subject to reiteration, as described in Example 19.As can be seen, iteration of the biotin tyramine treament results in a2.5 fold shift in the average fluorescence level of the cells.

(Events plotted against FL1LOG)

FIGS. 6(a)-(b) show the results of flow cytometry experiments describedin Example 20. (Events against FL1LOG.)

FIG. 6(a) shows results with mononuclear cells from blood labelled withanti-CD36.

FIG. 6(b) shows results for control enrichment, no CD36 antibody addedat the start.

FIG. 6(c) shows results for enriched cells labelled with anti-CD36;

FIG. 7 shows the results of experiments described in Example 21. Phagerecovered (×10⁵) is plotted for various concentrations ofbiotin-tyramine in μg/ml.

FIG. 8 shows nucleotide and amino acid sequences for the human homologueof the rat gene CL-6 identified for the first time in the work describedin Example 16. EcoRI cloning sites are underlined.

For one specific embodiment of the present invention, the procedure maybe summarised as follows, for purposes of illustration.

The exemplary system is based upon the use of immobilised reporterenzyme to catalyse the deposition of multiple copies of biotinylatedtyramine molecules around the site of enzyme activity. Catalysed enzymereporter deposition (CARD) has been used as a means of signalamplification in immunocytohemistry, ELISA and blotting formats (Bobrowet al. (1989) J. Immunol. Methods, 125: 279-295). The invention herecomes from the realisation that the deposition of a reporter moleculecan be used not only as an amplification system, but also as a transfersystem which allows recovery of tagged ligands.

In the example described here, horseradish peroxidase (HRP) activity isused to catalyse biotinylated tyramine molecule deposition. HRP activityis targeted to a specific site of interest, e.g. on a cell surface, bythe use of a primary mouse Mab with a desired binding specificity, theHRP activity being provided by an anti-mouse-HRP conjugated secondantibody which recognises the primary Mab. HRP activity mayalternatively be provided by direct conjugation of the Mab or ligand tothe enzyme (e.g. by expression as a fusion protein). Phage particlesdisplaying antibody antigen binding domains are incubated on the cellsurface along with the primary Mab, and those binding around the site ofthe primary Mab, and hence around the site of HRP activity, becomecovalently linked to biotin tyramine molecules. This reaction iscatalysed by oxygen free radicals generated by the HRP in the presenceof H₂O₂ (FIG. 1).

Biotinylated phage may then be specifically recovered using streptavidincoated magnetic beads and hence phage which bind in close proximity tothe existing mouse Mab are enriched for. The half life of thebiotin-tyramine phenolic free radical is very short, so depositionoccurs extremely close to the activating enzyme (Bobrow et al., supra).When CARD is used as an amplification system to enhance signal inimmunocytochemistry no detectable loss of image resolution is apparent,indicating that deposition occurs in close association with thecatalytic enzyme (Adams, J. C. (1992) J. Histochem. and Cytol. 40:1457-1463). The area over which the signal transfer occurs may beincreased or decreased by modifying the viscosity or temperature of thesolution in which the reaction is carried out, or by adding excessunbiotinylated tyramine.

Signal transfer selection has general applications to the identificationof protein-protein interactions and in some ways is analogous to theyeast two-hybrid system which has proved to be a very powerful techniquefor the detection of such interactions (Fields and Song, 1989, Nature340, 245-246). Both systems involve a tagged known protein which can bepaired with a library of unknown proteins, some of which may interactwith the tagged protein. Interaction between the two proteins in the twohybrid system results in transcriptional activation of the yeastGAL1-lacZ gene which encodes enzymes for galactose utilisation and henceallows selection of the interacting clone on galactose-containing media.Interaction of the two proteins in the signal transfer system results inlabelling of the unknown protein, e.g. phage-displayed antibody, peptideor other protein and hence recovery of that moiety. If phage-displayedantibody, peptide or other protein is the labelled (e.g. biotinylated)element then rescue of the gene for the interacting protein isfacilitated, since in phage display each phage particle contains nucleicacid encoding the antibody, peptide or other protein it displays (seee.g. WO92/01047). Signal transfer selection is not confined tointracellular expression in yeast, and as such has many advantages overthe yeast two-hybrid system.

Examples 15 and 16 demosntrate how signal transfer selection may be usedas a tool for discovering novel protein-protein interactions. Examplesof the types of protein-protein interactions which may be identifiedinclude proteins interacting in signal transduction pathways, such as Gproteins, kinases, phosphatases. Receptors often exist as multiproteincomplexes, interacting pairs of which may be identified either in thepresence or absence of ligand binding. Protein-protein interactionswhich occur within the cell may also be identified, for instance usingcell extracts, inside-out vesicles, nuclear extracts and extracts fromother cellular compartments, either in solution or immobilised on asolid support. The present invention may also be applied to theidentification of protein-DNA interactions. Segments of DNAencorporating putative transcription factor binding domains may belabelled (e.g. biotinylated) and coupled to enzyme-associated bindingmolecule for the label (e.g. streptavidin). Proteins which bind the DNAsequence may be selected by signal transfer selection.

There are many applications of signal transfer selections, which will beevident to people skilled in the art. Applications include the isolationof antibodies which specifically recognise a ligand-receptor complex,using an enzyme conjugate ligand to target the selection of suchantibodies. Specific labelling of one cell type over and abovebackground cell types may be achieved. For example, cells expressing oneparticular surface antigen may be labelled using an enzyme-conjugatedsbp member which recognises that antigen and which can transfer label tothose cells alone. This allows purification of the antigen-expressingcell type from a background of cells which do not express the antigenand do not, therefore, become labelled (or not significantly so). Thisis exemplified in Example 20.

Signal transfer labelling need not be limited to cell surfaces. Anyprotein, virus particle or other species in a complex mix may belabelled specifically and purified away from the unlabelled population.

Signal transfer has applications to signal enhancement in flowcytometry, as discussed and demonstrated in Examples 18 and 19. Thesignal enhancement profile may be used for particular molecules, e.g. ona cell surface, to assess copy number of that molecule, e.g. on aparticular cell or cell type, or to asess the proximity of two or moredifferent target molecules, e.g. on the same cell, as well as providinga more sensitive method for detection of a particular protein, e.g. on acell surface.

Another application is that of reverse drug screening. In this process adrug which is known to be efficacious, but the cellular target of whichis unknown, may be conjugated to the enzyme which directs labeldeposition. The drug-enzyme complex may then be incubated with cellularextracts and the labelling molecule added. Proteins in the cellularextract which bind to the drug-enzyme conjugate then become labelled,allowing for their purification and characterisation.

Since the signal transfer selection mechanism relies on the generationof free radicals use may be made of the generation of free radicals by aprotein or putative enzyme to select for a protein with novel orenhanced catalytic activity. Phage libraries of proteins, enzymes, orputative catalytic antibodies may be made and selection may be directedby the labelling (e.g. biotinylation) of active species due to theirability to generate free radicals which activate the label (e.g. biotintyramine) and cause its deposition on the phage displayed species.

Signal transfer technology also has a number of in vivo applications,for example in tumour targeting. An antibody-HRP conjugate whichspecifically recognises a tumour type may be allowed to localise to thetumour in vivo. Biotin tyramine, or a similar molecule, may then beinjected, and the HRP may catalyse biotin tyramine depositionspecifically at the tumour site. This would result in a heavilybiotinylated tumour to which streptavidin-conjuagte drugs, orstreptavidin-liposomes as vechicles for gene therapy or drug delivery,may be targeted.

Signal transfer is a process which can be re-iterated resulting in thesuccessive build up of biotin tyramine molecules around a focus ofenzyme activity. This may have In vivo applications e.g. in the contextof arteriole or nerve repair since successive layers of biotin tyramine,or similar molecules, may be depositied at sites of damage to generatecomplexes which may block damaged vessels.

The iterative potential of biotin-tyramine and other label despositionin accordance with the present invention may be used in the generationof oriented surfaces. Successive layers of proteins, or other species,may be deposited on the surface. An initial protein, or other species,may be immobilised on a surface and a binding molecule specific for thisinitial protein may be enzyme-(e.g. HRP-) conjugated and allowed to bindto the surface, then used to deposit a layer of biotine tyramine overthe initial surface. A second, e.g., streptavidin-linked protein, orother species, may then be added to the surface, giving a layer of thesecond protein. This process may be re-iterated as required to build upcomplex oriented layers on surfaces.

A model system has been used to exemplify the potential of thisinvention utilising a HeLa cell line which has been transfected with thegene for human carcinoembryonic antigen (CEA). A scfv which specificallyrecognises CEA has been used for initial experiments and a large scFvphage display library has been used to generate further anti-CEAspecific scFv's using the signal transfer selection system.

Further experiments have been carried out to select for specific cellsurface proteins on cultured human endothelial cells.

LIST OF EXAMPLES

EXAMPLE 1—Recovery of CEA-binding phage from the surface of cellsexpressing CEA in the presence or absence of a marker anti-CEA mouseantibody.

EXAMPLE 2—Selection of human CEA-binding phage from a large library ofhuman scFv's.

EXAMPLE 3—K_(off) determination for scFv fragments binding to CEA.

EXAMPLE 4—Selection of phage which bind to the mouse anti-CEA antibodyfrom a large library of human scFv's.

EXAMPLE 5—Marker-ligand-dependent biotinylation of a CEA-expressing celltype.

EXAMPLE 6—Marker-ligand dependent biotinylation of CEA.

EXAMPLE 7—Selection of human E-selectin-binding phage from a largelibrary of human scFv's.

EXAMPLE 8—Selection of novel anti-TGFβ1-binding phage using an existinganti-TGFβ1-specific scFv.

EXAMPLE 9—Selection of anti-chemokine receptor phage using a chemokineligand to guide selection.

EXAMPLE 10—Selection of anti-chemokine receptor phage using-light-activated streptavidin and the receptor ligand to guide.

EXAMPLE 11—Selection of phage antibodies to two different cell surfaceadhesion molecules using a biotinylated ligand which binds to both toguide selection.

EXAMPLE 12—Measurement of the distance over which signal transfer usingbiotin tyramine may occur.

EXAMPLE 13—Step-back selection to isolate phage antibodies which inhibitligand binding.

EXAMPLE 14—Biotin tyramine selection In solution using a peptide phagelibrary.

EXAMPLE 15—Characterisation of clones which bind to CD4+ cells, but notto the chemokine receptor CC-CKR5, by Western blotting and ICC.

EXAMPLE 16—Demonstration of the use of signal transfer selection toidentify novel protein-protein interactions.

EXAMPLE 17—Biotinylation of CD4E1 phage on the cell surface using MIP-1αto direct the biotinylation.

EXAMPLE 18—Use of biotin tyramine as a signal amplification reagent inflow cytometry.

EXAMPLE 19—Iteration of biotin tyramine treatment to give further signalenhancement.

EXAMPLE 20—Use of biotin tyramine to specifically biotinylatesubpopulations of cells to allow their subsequent purification.

EXAMPLE 21—Biotinylation of phage particles in solution to validatebiotin-tyramine preparations.

EXAMPLE 1 Recovery of CEA-Binding Phage from the Surface of CellsExpressing CEA in the Presence of a Marker Anti-CEA Mouse Mab.

a. Purification of CEA-binding phase

CEA6 is a CEA specific scFv isolated from a large scFv phage displaylibrary by panning on human CEA (Vaughan et al 1996). OP1 is a controlscFv which recognises a 16 residue peptide and does not bind to CEA.Phagemid particles expressing CEA6 or OP1 scFv's as a fusion proteinswith the phage gIII protein were isolated as follows. 500 ml prewarmed(37° C.) 2YTAG (2TY media supplemented with 100 μg/ml ampicillin and 2%glucose) in a 2 l conical flask was inoculated with approximately 3×10¹⁰cells from a glycerol stock (−70° C.) of CEA6- or OP1-phagemid. Theculture was grown at 37° C. with good aeration until the OD 600 nmreached 0.8. M13K07 helper phage (Stratagene) was added to the cultureto a multiplicity of infection (moi) of approximately 10 (assuming thatan OD 600 nm of 1 is equivalent to 5×10⁸ cells per ml of culture. Theculture was incubated stationary at 37° C. for 15 minutes followed by 45minutes with light aeration (200 rpm) at the same temperature. Theculture was centrifuged and the supernatant drained from the cellpellet. The cells were resusupended in 500 ml 2TYAK (2YT mediasupplemented with 100 μg/ml ampicillin and 50 mg/ml kanamycin), and theculture incubated overnight at 30° C. with good aeration (300 rpm) Phageparticles were purified and concentrated by three polyethylene glycol(PEG) precipitations (Sambrook, J., Fritsch, E. F., and Maniatis, T.(1990). Molecular Cloning—A Laboratory Manual. Cold Spring Harbour,N.Y.) and resuspended in PBS to 10¹² transducing units (tu)/ml.

b. Preparation of HeLa-CEA cell slides.

CEA-expressing HeLa cells were grown to confluence in DMEM supplementedwith 10% fetal calf serum on 16 chamber slides (Nunc). The cells werefixed with acetone for 10 minutes, dried and stored at −70° C.

c. Biotinylation of tyramine.

An equimolar amount of tyramine (Sigma) was allowed to react withNHS-LC-biotin in 50 mM borate buffer, pH 8.8. The reaction was carriedout at room temperature overnight in the dark with rotation. Thebiotinylated tyramine (BT) was filtered through a 0.45 μM filter,aliquotted and stored at −70° C.

d. Biotinylation of phage binding in close proximity to the Mab.

HeLa-CEA slides were incubated overnight at 4° C. with 100 μl phage inthe presence or absence of an anti-CEA mouse Mab (Zymed) at a range ofdilutions from 1:100 to 1:10000 in 3% marvel PBS (MPBS). Phage imputvalues were around 5×10¹¹ per ml for CEA-purified phage. Controlincubations were carried out in parallel using a phage preparation ofOP1 in the presence of the anti-CEA Mab. 100 μl of phage were used perchamber of the slide. Slide chambers were washed 3 times in PBScontaining 0.1% Tween® 20 (polyoxyethylenesorbitan monolaurate) (PBST),followed by 3 washes with PBS. Each wash was left for 2 minutes beforebeing changed. 100 μl of a goat anti-mouse HRP second antibody (Pierce)was then added at a dilution in MPBS of 1:2500 and incubated for 1 hourat room temperature. Control incubations were carried out for the samelength of time incubating with PBS alone. Washing was carried out asbefore and 100 μl of BT in 50 mM Tris-HCl pH 7.4 with 0.03% H₂O₂ wasadded to each slide chamber for 10 minutes at room temperature. Controlincubations were carried out as above, but with the omission of the BT.Chambers were washed as above and phage were then eluted using 200 μltriethylamine (TEA). TEA was neutralised with 100 μl of 1M Tris-HCl pH7.4. 10 μl of this eluted phage was used to directly infect anexponentially growing culture of E coli TG1. Infected cells were grownfor 1 hour at 37° C. with light aeration in 2YT broth, and then platedon 2TYAG medium. A series of dilutions of bacteria were plated out andincubated at 30° C. overnight. Colony counts gave the phage titre. Theresults are shown in Table 1.

e. Capture of biotinylated phage on streptavidin-coated magnetic beads.

20 μl of streptavidin-coated magnetic beads (Dynal) were taken out ofsolution using a magnet and blocked for 2 hours at room temperature on arotating platform with 1 ml of 3% MPBS. Beads were pelleted using amagnet and 150 μl of eluted phage with 30 μl of 15% MPBS were then addedto the blocked beads and rotated for 15 minutes at room temperature.Beads were pelleted, washed 3 times in PBST and 3 times in PBS. Thebeads were resuspended in a final volume of 100 μg PBS. Half of this wastaken and used to directly infect 1 ml of an exponentially growingculture of E coli TG1. Infected cells were grown for 1 hour at 37° C.with light aeration in 2YT broth, and then plated on 2TYAG medium. Aseries of dilutions of bacteria were plated out and incubated at 30° C.overnight. Colony counts gave the phage titre. The results are shown inTable 1.

2. Summary of the results—enhanced recovery of CEA-binding phage usingsignal transfer selection followed by streptavidin capture.

Incubations of CEA6 purified phage on slides coated with CEA transfectedHeLa cells were carried out under a range of different conditions. Phageimput, primary Mab dilution, presence or absence of HRP-conjugatedsecond antibody and presence or absence of BT were all examined. OP1, anon-CEA-specific phagemid which had been selected on a 16 residuepeptide was also included. The data are shown in Table 1.

CEA6 phage incubated in the presence of primary Mab, anti-mouse-HRPconjugated second antibody and BT consistently gave the highest numberof phage recovered on the streptavidin-coated magnetic beads. When BTwas omitted the number of phage recovered fell by 16-fold, and when theprimary Mab was omitted phage recovery was reduced by 8-fold. Absence ofthe HRP conjugated Mab resulted in a 6-fold reduction in phage recoverysupporting the conclusion that biotinylation of CEA6 phage is driven bythe presence of the Mab-HRP complex. This also demonstrates that only asmall proportion of phage are binding non-specifically to the Dynalbeads in the absence of BT. Some non-site-specific biotinylation ofphage must be occurring since the recovery of phage in the presence ofBT, but absence of primary Mab is greater than the recovery when BT isomitted. Absence of the HRP-antibody conjugate has a simlar effect onthe number of phage recovered compared with absence of the primary Mab.This suggests that the secondary Mab is binding specifically to theprimary Mab and gives little background binding to the cells themselves.The non-CEA-specific phage gave similar levels of biotin-phage recoveryas those seen in the absence of the primary anti-CEA Mab, againsuggesting a low level of non-site-specific phage biotinylation.

Overall the results provide an exemplary demonstration of how anexisting Mab raised to a protein of interest can be used to guidecatalysis of biotin deposition onto phage binding the protein ofinterest in the same vicinity as that Mab.

EXAMPLE 2 Selection of CEA-Binding Phage from a Large Library of HumanSCFV'S

Antibody repertoire

The following antibody repertoire was used. Large single chain Fvlibrary derived from lymphoid tissues including tonsil, bone marrow andperipheral blood lymphocytes.

Polyadenylated RNA was prepared from the B-cells of various lymphoidtissues of 43 non-immunised donors using the QUICKPREP® (mRNA extractionkit; Pharmacia). First-strand cDNA was synthesized from mRNA using a“First-strand cDNA synthesis” kit (Pharmacia) using random hexamers toprime synthesis. V-genes were amplified using family-specific timers forVH, Vκ and Vλ genes as previouslydescribed (Marks et al., (1991) J. Mol.Biol. 222:581-597) and subsequently recombined together with the (Gly₄,Ser)₃ scFv linker by PCR assembly. The VH-linker-VL antibody constructswere cloned into the Sfi I and Not I sites of the phagemid vector,pCANTAB 6. Ligation, electroporation and plating out of the cells was asdescribed previously (Marks et al, supra). The library was made ca.1000× larger than that described previously by bulking up the amounts ofvector and insert used and by performing multiple electroporations. Thisgenerated a scFv repertoire that was calculated to have ca. 1.3×10¹⁰individual recombinants which by Bst NI fingerprinting were shown to beextremely diverse.

a. Induction of phage antibody library

The phage antibody repertoire above was selected for antibodies to CEA.The ‘large’ scFv repertoire was treated as follows in order to rescuephagemid particles. 500 ml prewarmed (37° C.) 2YTAG (2YT mediasupplemented with 100 μg/ml ampicillin and 2% glucose) in a 2 l conicalflask was inoculated with approximately 3×10¹⁰ cells from a glycerolstock (−70° C.) culture of the library. The culture was grown at 37° C.with good aeration until the OD600 nm reached 0.7 (approximately 2hours). M13K07 helper phage (Stratagene) was added to the culture to amultiplicity of infection (moi) of approximately 10 (assuming that anOD600 nm of 1 is equivalent to 5×10⁸ cells per ml of culture). Theculture was incubated stationary at 37° C. for 15 minutes followed by 45minutes with light aeration (200 rpm) at the same temperature. Theculture was centrifuged and the supernatant drained from the cellpellet. The cells were resuspended in 500 ml 2YTAK (2YT mediasupplemented with 100 μg/ml ampicillin and 50 μg/ml kanamycin), and theculture incubated overnight at 30° C. with good aeration (300 rpm).Phage particles were purified and concentrated by three polyethyleneglycol (PEG) precipitations (Sambrook, J., Fritsch, E. F., & Maniatis,T. (1990). Molecular Cloning—A Laboratory Manual. Cold Spring Harbour,N.Y.) and resuspended in PBS to 10¹² transducing units (tu)/ml(ampicillin resistant clones).

b. Selection of CEA-binding phase from a large non-immunised phasedisplay library using catalysed enzyme reporter deposition followed bystreptavidin capture.

i. First round of selection

Two rounds of selection using phage prepared from a large non-immunisedhuman scFv library were carried out on slides of CEA-expressing HeLacells. 5×10¹¹ phage were allowed to bind to the cells in the presence orabsence of an anti-CEA mouse Mab (Zymed) at a dilution of 1:100 in MPESin a total volume of 100 μl, at 4° C. overnight. Slides were washedthree times in PBST followed by three times in PBS. A secondaryanti-mouse hydrogen-peroxidase-conjugated antibody which recognised theprimary mouse anti-CRA antibody was then incubated on the sections at adilution of 1:2500 in MPBS in a total volume of 100 μl at roomtemperature for 1 hour. Washing was carried out as before and 100 μl ofbiotinylated-tyramine in 50 mM Tris-HCl pH 7.4 with 0.03% H₂O₂ was addedto each slide chamber for 10 minutes at room temperature. Chambers werewashed as above and phage were eluted using 200 μl triethylamine (TEA).TEA was neutralised with 100 μl of 1M Tris-HCl pH 7.4.

ii. Assessment of the total number of phage binding to the HeLa-CEAcells

10 ml of this eluted phage was used to directly infect an exponentiallygrowing culture of E coli TG1 with light aeration in 2TY broth at 37° C.for 1 hour. Infected TG1s were plated on 2TYAG medium in 243 mm×243 mmdishes (Nunc). Dilutions of infected TG1s were also plated out andincubated at 30° C. overnight. Colony counts gave the phage outputtitre.

iii. Recovery of biotinylated phage on streptavidin-coated magneticbeads

20 μl of streptavidin-coated magnetic beads (Dynal) were taken out ofsolution using a magnet and blocked for 2 hours at room temperature on arotating platform with 1 ml of 3% MPBS. Beads were pelleted and 150 μlof eluted phage with 30 μl of 15% MPBS were then added to the blockedbeads and rotated for 15 minutes at room temperature. Beads werepelleted, washed 3 times in 1 ml PBST and 3 times in 1 ml PBS. The beadswere resuspended in a final volume of 100 μl PBS. 50 μl of this wastaken and used to directly infect 1 ml of an exponentially growingculture of E. coli TG1 at 37° C. for 1 hour with light aeration in 2TYAGmedium. Infected TG1s were plated on 2TYAG medium in 243 mm×243 mmdishes (Nunc). Dilutions of bacteria were also plated out and incubatedat 30° C. overnight. Colony counts gave the phage output titre.

iv. Second round of selection

Colonies were scraped off the 243 mm×243 mm plates into 3 ml of 2TYbroth and 15% (v/v) glycerol added for storage at −70° C. Glycerol stocksolutions from the first round of selection of the repertoire on theHeLa-CEA cells were rescued using helper phage to derive phagemidparticles for the second round of selection. Phagemid particles wererescued from both first round selections carried out in the presence orin the absence of the marker anti-CEA Mab. 250 μl of glycerol stock wasused to inoculate 50 ml 2YTAG broth, and incubated in a 250 mL conicalflask at 37° C. with good aeration until the OD600 nM reached 0.7(approximately 2 hours). M13K07 helper phage (moi=10) was added to theculture which was then incubated stationary at 37° C. for 15 minutesfollowed by 45 minutes with light aeration (200 rpm) at the sametemperature. The culture was centrifuged and the supernatant drainedfrom the cell pellet. The cells were resuspended in 50 ml prewarmed2YTAK, and the culture incubated overnight at 30° C. with good aeration.Phage particles were purified and concentrated by PEG precipitation(Sambrook et al., 1990) and resuspended in PBS to 10¹³ tu/ml.

Phage recovered from the selection in the presence of the anti-CEA mouseMab underwent a second round selection with either no Mab, or with a1:100, or a 1:1000 dilution of the anti-CEA Mab. Phage recovered fromthe first round of selection in the absence of the anti-CEA Mabunderwent a second round of selection, again in the absence of theanti-CEA Mab. The selections were carried out on the HeLa-CEA cells asdescribed for the first round of selection. The total numbers of phagepresent in the eluates and recovered by streptavidin capture are shownin Table 2.

The total number of phage recovered on the magnetic beads after thefirst round of selection was comparable either in the presence orabsence of Mab. At round two of the selection the total number ofrecovered phage had dropped to around one tenth of the value from roundone. It was, however, notable that the number of phage recovered aftertwo rounds of selection in the presence of Mab was around 7-fold higherthan that recovered after two rounds of selection without the Mab beingpresent. When one round with Mab present was followed by one roundwithout the Mab the number of recovered phage was around half of thatseen after two rounds of selection with the Mab. Ten-fold dilution ofthe Mab at round 2 of the selections slightly reduced the number ofphage recovered on the Dynal beads (by 12%)

c. Growth of single selected clones for immunoassay

Individual colonies from the first and second round selections were usedto inoculate 100 μl 2YTAG into individual wells of 96 well tissueculture plates (Corning). Plates were incubated at 30° C. overnight withmoderate shaking (200 rpm). Glycerol to 15% was added to each well andthese master plates stored at −70° C. until ready for analysis.

d. Soluble ELISA to identify anti-CEA scFv

Cells from the master plates were used to inoculate fresh 96 well tissueculture plates containing 100 μl 2YTAG per well. These plates wereincubated at 30° C. for 8 hours then centrifuged at 2000 rpm for 10 minand the supernatant eluted. Each cell pellet was resuspended in 100 μl2YTA containing 10 mM IPTG and incubated at 30° C. overnight.

Each plate was centrifuged at 2000 rpm and the 100 μl supernatant fromeach well recovered and blocked in 20 μl 18% M6PBS stationary at roomtemperature for 1 hour. Meanwhile, flexible microtitre plates which hadbeen blocked overnight stationary at 37° C. with either 100 μl 0.5 μg/mlCEA in dH₂O or 100 μl dH₂O alone, were washed 3 times in PBS and blockedfor 2 h stationary at room temperature in 3MPBS. These plates were thenwashed three times with PBS and 50 μl preblocked soluble scFv added toeach well of both the CEA-coated or uncoated plate. The plates wereincubated stationary at 37° C. for 1 h after which the scFv solutionswere poured off. The plates were washed by incubating for 2 min in PBSTthree times followed by incubating for 2 min in PBS three times, all atroom temperature.

To each well of both the CEA-coated and the uncoated plate, 100 μl of a1 in 200 dilution of the anti-myc tag murine antibody 9E10 (Munro, S. &Pelham, H. R. B. (1986) Cell 46, 291-300) in 3MPBS was added and theplates incubated at 37° C. stationary for 1 h. Each plate was washed asdescribed above and 100 μl of a 1 in 5000 dilution goat anti-mousealkaline phosphatase conjugate (Pierce) in 3MPBS added and incubatedstationary at 37° C. for 1 h. Plates were washed as described abovefollowed by two rinses in 0.9% NaCl. Alkaline phosphatase activity wasvisualised using the chromagenic substrate pNPP (Sigma). The absorbancesignal generated by each clone was assessed by measuring the opticaldensity at 405 nm (pNPP) using a microtitre plate reader. Clones werechosen for further analysis if the ELISA signal generated on theCEA-coated plate was at least double that on the uncoated plate. Thenumber of clones screened from each round of selection and the number ofCEA positives are shown in Table 3.

e. Sequencing of anti-CEA ScFv Antibodies

The nucleotide sequences of the anti-CEA antibodies were determined byfirst using vector-specific primers to amplify the inserted DNA fromeach clone. Cells from an individual colony on a 2YTAG agar plate wereused as the template for a polymerase chain reaction (PCR) amplificationof the inserted DNA using the primers pUC19reverse and fdtetseq.Amplification conditions consisted of 30 cycles of 94° C. for 1 min, 55°C. for 1 min and 72° C. for 2 min, followed by 10 min at 72° C. The PCRproducts were purified using a PCR Clean-up Kit (Promega) in to a finalvolume of 50 μl H2O. Between 2 and 5 μl of each insert preparation wasused as the template for sequencing using the Taq Dye-terminator cyclesequencing system (Applied Biosystems). The primers mycseq10 andPCR-L-Link were used to sequence the light chain of each clone andPCR-H-Link and pUC19reverse to sequence the heavy chain.

f. Sequence of the initial CEA-specific scFv antibodies

Twelve different CEA specific antibodies were isolated from theselections. Each clone name and its heavy and light chain germ line isgiven below. The signal transfer method of selection is capable ofgenerating a diverse panel of anti-CEA antibodies. None of theseantibodies were isolated from experiments in which panning of the largescFv library was carried out directly on purified CEA, suggesting thatsignal transfer selection provides a way of accessing different antibodyspecificities from the library.

CLONE VH GERMLINE VL GERMLINE SS1A4 VH4 DP71 VLambda2 DPL11 SS1A11 VH4DP71 VLambda2 DPL11 SS1G12 VH4 DP71 VKappa1 L12 SS22A4 VH4 DP79 VLambda1DPL5/2 SS22A9 VH4 DPG3 VLambda3 DPL16 SS22B7 VH4 DP79 VLambda1 DPL5/2SS22B1 VH2 V11-5b VLambda1 DPL2 SS22D12 VH3 V343 VLambda1 DPL2 SS22E4VH2 DP28 Vkappa1 DPK8 SS21B1 VH4 DP70 Vkappa1 DPK4 SS21B7 VH1 DP71Vlambda3 DPL16 SSDS1 VH4 DP78 Vlambda3 DPL16

EXAMPLE 3 K_(OFF) Determination for SCFV Fragments Binding toDesialylated CEA

a. K_(off) determination by surface plasmin resonance

The K_(off)'s for binding to CEA of the scFv fragments described inExample 2 were determined using desialylated CEA coupled to a CM5 sensorchip. 100 μg of CEA was resuspended in 0.1M sodium acetate buffer pH 4.0and desialylated using 1.375 mU sialidase (Sigma). This was incubatedfor 4 hours at 37° C. with occasional shaking. The desialylated CEA wasthen oxidised using 1 unit of galactose oxidase per 500 μg of CEA in 10mM phosphate buffer pH7.0. This was incubated for 2 hours at 36° C. anddesalted into 10 mM sodium acetate buffer pH4.0. The CEA was thenimmobilised onto the sensor chip using the aldehyde group. 15 μl EDC/NHScoupling agent (Pierce) was passed over the chip at a flow rate of 5μl/min. 35 μl of 5 mM hydrazine in water was then passed over the chip,followed by 35 μl of ethanolamine. 4 μl of 60 μg/ml treated CEA waspassed over the chip at a flow rate of 2 μl/min followed by 40 μl of0.1M sodium cyanoborohydride in 0.1M acetate buffer pH4.0 at a flow rateof 5 μl/min. Approximately 1500 RU (resonance units) of CEA was boundusing this method. 5000 RU and 800 RU CEA chips were made using thisprocedure.

K_(off)'s were calculated using the Bia-Evalution software(Pharmacia)—Saturation of the chip with purified scFv was demonstratedfor each sample before K_(off) was measured. Results are shown in table4. The range of K_(off)'s of the selected antibodies suggests thatrecovery is dependent on the exact site of binding of the phageantibodies rather than the affinity of the interaction, as is the casewith traditional selection methods. Signal transfer selection is,therefore, a route to obtaining a population of antibodies of diversesequences and affinities which would not normally be obtained by otherselection procedures.

EXAMPLE 4 Selection of Phage Which Bind to the Mouse Anti-CEA Antibodyfrom a Large library of Human SCFV'S

The antibody repertoire used here and the method of phage induction wasthe same as that described in Example 2. The selections assayed were thesame as those described in Example 2.

a. Growth of single selected clones for immunoasaay

Individual colonies from the first and second round selections were usedto inoculate 100 μl 2YTAG into individual wells of 96 well tissueculture plates (Corning). Plates were incubated at 30° C. overnight withmoderate shaking (200 rpm). Glycerol to 15% was added to each well andthese master plates stored at −70° C. until ready for analysis.

b. Soluble ELISA to identify anti-scFv

Cells from the master plates were used to inoculate fresh 96 well tissueculture plates containing 100 μl 2YTAG per well. These plates wereincubated at 30° C. for 8 hours then centrifuged at 2000 rpm for 10 minand the supernatant eluted. Each cell pellet was resuspended in 100 μl2YTA containing 10 mM IPTG and incubated at 30° C. overnight.

Each plate was centrifuged at 2000 rpm and the 100 μl supernatant fromeach well recovered and blocked in 20 μl 18% M6PBS stationary at roomtemperature for 1 hour. Meanwhile, flexible microtitre plates which hadbeen incubated overnight stationary at 37° C. with either 50 μl 0.1μg/ml of anti-CEA mouse Mab in PBS or 50 μl PBS alone, were washed 3times in PBS and blocked for 2 h stationary at room temperature in3MPBS. These plates were then washed three times with PBS and 50 μlpreblocked soluble scFv added to each well of both the anti-CEA-mouseMab-coated or uncoated plate. The plates were incubated stationary at37° C. for 1 h after which the sCFV solutions were poured off. Theplates were washed by incubating for 2 min in PBST three times followedby incubating for 2 min in PBS three times, all at room temperature.

To each well of both the mouse Mab-coated and the uncoated plate, 100 μlof a 1 in 200 dilution of biotinylated anti-myc tag murine antibody 9E10(Munro, S. & Pelham. H. R. B. (1986) Cell 46, 291-300) in 3MPBS wasadded and the plates incubated at 37° C. stationary for 1 h. Each platewas washed as described above. Plates were then incubated withalkaline-phosphatase-streptavidin complex (DAKO) diluted 1:1000 in dH₂O.Plates were washed as described above followed by two rinses in 0.9%NaCl. Alkaline phosphatase activity was visualised using the chromogenicsubstrate pNPP (Sigma). The absorbance signal generated by each clonewas assessed by measuring the optical density at 405 nm (pNPP) using amicrotitre plate reader. Clones were scored for positive binders for theanti-CEA mouse Mab if the ELISA signal generated on the CEA-coated platewas at least double that on the uncoated plate.

Clones from the various round 2 selections were screened for anti-CEAmouse Mab binding (Table 5). 12.5% of clones which had come through tworounds of 1:100 Mab selections were found to bind the Mab. No Mabbinders were present in the population which came through two rounds ofselection with no Mab present. This demonstration that some of therecovered phage recognise the anti-CEA mouse Mab is evidence for generalbiotinylation of any phage binding in close proximity of the anti-mouseHRP secondary antibody and hence is evidence of the site-specific natureof the interaction.

EXAMPLE 5 Marker-Ligand-Dependent Biotinylation of a CEA-Expressing CellType

a. Biotinylation by biotin tyramine of the HeLa-CEA expressing cellsgrown on slides.

Thawed HeLa-CEA slides which had been rehydrated in PBS for 10 minutesat room temperature were incubated for 15 miinutes with streptavidin inPBS at 5 μg/ml. Slides were washed four times in PBS and then incubatedfor 15 minutes with in PBS at 10 μg/ml. Slides were washed 4 times inPBS and then incubated in block consisting of 1% BSA PBS containing 10%normal mouse serum for 30 minutes. Block was removed and the slides thenincubated with CEA6 purified phage (as described in Example 1) atapproximately 1×10¹⁰ per ml in 1% PBS-BSA overnight at 4° C. Controlslides were incubated under the same conditions with purifiedfluorescein-binding phage which do not recognise CEA. Slides were movedback to room temperature and washed in PBST for 10 minutes, followed byincubation with 9E10-biotin at 3 μg/ml diluted in 1% BSA-PBS for 1 hour.Washing was carried out for 10 minutes in PBST and the slides thenincubated with ABC-HRP (DAKO) diluted 1:100 in PBS for 30 minutes.Slides were either developed at this point or washed three times inPBST, then incubated in either with biotinylated tyramine in 50 mMTris-HCl pH 7.4 containing 0.03% H₂O₂ for 10 minutes. Three PBST washeswere carried out and the slides then incubated with the ABC-HRP complexagain for 30 minutes. Slides were developed using carbazole. Carbozolewas prepared freshly by dissolving 9-amino-ethyl-carbozole (Sigma) at 60mg per 25 ml DMF then adding 100 μl of this to 1 ml sodium acetatepH5.2. 5 μl of 30% H₂O₂ was then added and 100 μl of this mix added toeach slide chamber. Development was left for 20 minutes and then thecarbazole washed off with dH₂O.

Slides incubated with the CEA6 phage but without the biotin-tyramineamplification step showed faint red staining in regions blebbing fromthe HeLa-CEA cell surfaces, whereas the slides treated with theanti-fluorescein phage showed no such staining. Slides incubated withCEA6 phage and then subjected to a round of biotin tyramine treatmentshown significantly stronger staining of the regions of CEA,demonstrating that proteins present in the region of CEA6 phage bindinghad been biotinylated and were able to amplify the colour reaction dueto recruitment of more ABC-HRP complex.

EXAMPLE 6 Marker-Ligand Dependent Biotinylation of CEA

i. Biotinylation of CEA.

HeLa-CEA expressing cells grown in chamber flasks were blocked in MPBSfor 2 hours at room temperature. 100 μl of an anti-CEA mouse Mab wasthen incubated on the slides at a dilution of 1:100 in MPBS for 1 hourat room temperature. Control incubations were carried out in MPBSwithout the presence of the anti-CEA Mab. Slides were washed three timesin PBST followed by three washes in PBS. 100 μl of of a goat arti-mouseHRP-conjugated second antibody (Pierce) was then added at a dilutionof1:2500 in MPBS and incubated for 1 hour at room temperature. Washingwas carried out as before and 100 μl of biotinylated tyramine in 50 mMtris-HCl pH 7.4 with 0.03% H₂O₂ was added to each slide chamber for 10minutes at room temperature. Cells were then scraped off the slides.Cells were pelleted at 600 rpm for 5 minutes and then resuspended in 10mM triethanolamine, 1% triton in saline. Cells were left on ice for 10minutes, then cell nuclei were pelleted at 13000 rpm in a minifuge for 5minutes at 4° C. Supernatants were added to reducing protein loadingbuffer and run on 10-15% SDS gradient PHAST gels. Protein weretransferred to HYBOND C EXTRA® (membranes for binding proteins,Amersham) membranes using the PHAST system programme at 70° C. for 30minutes. Membranes were blocked for 2 hours in MPBS and incubated for 1hour at room temperature with either a strepavidin-HRP complex, or ananti-CEA mouse Mab. Blots probed with the anti-CEA mab were washed threetimes in PBST followed by three washes in PBS, then incubated with ananti-mouse-HRP-conjugated antibody at a diltuion of 1:2500 in MPBS for 1hour at room temperature. Blots were washed as before and developedusing the ECL (Amersham) detection kit.

The western blot probed with streptavidin-HRP conjugate showed thepresence of one major high molecular weight band in the Hela-CEA cellstreated with anti-CEA, anti-mouse-HRP and then biotinylated tyramine.This band was shown to be reactive with an anti-CEA Mab. The band was ahigher molecular weight than that theoretically anticipated for CEA,probably due to the many carbohydrate groups on CEA which result inretarded migration of the CEA. Two other biotinylated minor bands couldbe detected at round the expected size for a Mab or conjugated Mab.These bands could potentailly be biotinylated forms of the anti-CEA Maband the anti-mouse-HRP conjugate. No other clear bands could be seen onthe blot, although some some less specific biotinylation may beindicated by the presence of a high molcular weight smear after a longexposure (20 minutes) of the blot to ECL (Amersham) film in the lanecorresponding to the Hela-CEA cells which were treated with bothantibodies and the BT. Control lanes, in which treatment of the cellswith BT or with the anti-CEA Mab was omitted, showed no evidence ofbiotinylation. This demonstrates the ability of the biotin tyraminesystem to selectively “tag” proteins binding in close proximity to amarker ligand to allow their detection and facilitate theirpurification.

EXAMPLE 7 Selection of Human Anti-E-Selection-Binding Phage from a LargeSCFV Library

a. Conjugation of polyclonal anti-E selectin IgG to HRP

Polyclonal anti-human-E-selectin IgG was obtained from R and D Systems.The conjugation was carried out using a hydrogen peroxidase conjugationkit supplied by Pierce. 1 mg of maleimide-activated HRP was conjugatedto 100 μg of Mab using the SATA protocol (Pierce). 20 μl of a 4 mg/mlSATA solution made up in DMF was added to 100 μl of polyclonal IgG inPBS. This was incubated for 30 min at room temperature, then 100 μl ofdeacetylation solution (Pierce) was added and incubation was continuedfor a further 2 hours at room temperature. Deacetylated IgG wasseparated from unreacted and deacetylated SATA on a 5 ml sepharose 25column which had been pre-equilibriated with maleimide conjugationbuffer. 0.5 ml fractions were collected and the majority of the proteinwas collected in fractions 2 and 3. 1 ml of the deacetylated IgG wasthen added to 1 mg of maleimide-activated HRP and incubated at roomtemperture for 1 hour.

b. Cell culture

Human vascular endothelial cells (HUVECs) (Clonetics) were cultured topassage 3 on 24 well plates (Nunc) coated with 1% gelatin. The cellswere grown to approximately 80% confluence using EGM medium (Clonetics).HUVEC cells express a low basal level of the adhesion proteinE-selectin.

c. Selection procedure

Cells were washed with PBS and the cells were then incubated overnightat 4° C. in 200 μl of PBS/1% BSA in the presence of 1×10¹² phageprepared from a large non-immunised human scFv library. To one culturewell a 1:20 dilution of the HRP-conjugated polyclonal anti-E selectinIgG was also added to the phage. Cells were washed three times in 0.5 mlPBST and three times in PBS. 200 μl of the biotin tyramine mix (as inExample 2 part bi) was added to each well and left for 10 minutes atroom temperature. Cells were washed as before and the phage then elutedin 200 ml triethylamine (TEA) for 10 minutes at room temperature. TheTEA was then neutralised with 100 μl of 1M Tris HCl pH7.4.

d. Recovery of biotinylated phage

20 μl of streptavidin-coated magnetic beads (Dynal) were taken out ofsolution using a magnet and blocked for 2 hours at room temperature on arotating platform with 1 ml of 3% MPBS. Beads were pelleted and 300 μlof eluted phage with 60 μl of 15% MPBS were added to the blocked beadsand rotated for 15 minutes at room temperature. Beads were pelleted,washed three times in 1 ml PBST and three times in 1 ml PBS. The beadswere resuspended in a final volume of 100 μl PBS. 50 μl of this wastaken and used to directly infect 5 ml of an exponentially growingculture of E coli TG1 at 37° C. for 1 hour with light aeration in 2TYAGmedium. Infected TG1s were plated on 2TYAG medium in 243 mm×243 mmdished (Nunc). Dilutions of bacteria were also plated out and incubatedat 30° C. overnight. Colony counts gave the phage output titre.

Output titres for selections:

Total Captured % phage eluate phage captured Minus anti-E-sel-HRP 8 ×10⁴ 81 0.10 conjugate Plus anti-E-sel-HRP 1.6 × 10⁴   498 3.11 conjugate

The percentage of biotinylated phage captured on the beads in thepresence of the HRP-conjugated polyclonal anti-E selectin IgG is around30-fold higher than the percentage captured in the absence of theantibody. This suggests the HRP-anti-E-selectin polyclonal IgG istargeting the biotinylation of E-selectin-specific phage.

e. Growth of single selected clones for soluble ELISA to identifyanti-E-selectin scFv

Single colonies were grown up exactly as described in Example 2 part cand the ELISAs were carried out as in part d, except that the plateswere coated with 1 μg/ml recombinant E selectin (R and D Systems).

The number of positives screened from each round of selection and thenumber of B-selectin positive clones are shown below.

No. clones E-sel + ve % E-sel + ve Minus anti-E sel HRP 95 0 0 conjugatePlus anti-E sel HRP 282 8 2.8 conjugate

The ELISA results demonstrate the increase in the number of E-selectinbinders selected for in the presence of the polyclonal anti-E selectinHLP conjugate compared to the selection when this antibody is omitted.This demonstrates that the antibody-HRP conjugate is responsible for thespecific biotinylation of phage binding in close proximity to it.

EXAMPLE 8 Selection of Novel TGFβ1-Binding Phage using an ExistingAnti-TGFβ1-Specific scFv

31G9 is a high affinity (1.2×10⁻⁹ M) anti-TGFβ1-specific scFv which waspreviously isolated from a large human non-immunised scfv phage displaylibrary by direct selection of the library on immobilised TGFβ1. Theantibody does not recognise a neutralising epitope of TGFβ1.Investigations were carried out to assess whether a HRP-conjugate of31G9 could be used in a signal transfer selection to isolate newlineages of phage antibodies which recognise different, potentiallyneutralising epitopes of TGFβ1.

a. Conjugation of 31G9 scFv to HRP.

31G9 was conjugated to maleimide-activated HRP as described in Example7, part a, except that 300 μg of purified scFv was used in theconjugation reaction.

b. Preparation of a low density TGFβ1 BIACORE® (biosensor instrument,Pharmacia) chip.

50 μl of NHS/EDC reagent (Pharmacia) was incubated for 30 min at roomtemperature on the surface of a CM5 chip. The chip was washed 5 times inHBS and 75 ng of TGFβ1 in 75 μl of 10 mM sodium citrate buffer pH 3.6was then incubated on the chip for 1 hour at room temperature. The chipwas washed 5 times in HBS and then treated with 1M ethanolamine pH8 for10 min The chip was stored at 4° C. in HBS. Approximately 40 resonanceunits (Rus) of TGFβ1 were linked to the chip.

c. Selection procedure

i) First round of selection.

100 μl of HRP-conjugated 31G9 (approximately 30 μg) was incubated on theTGFβ1-coupled BIACORE® (biosensor instrument, Pharmacia) chip for 1 hourat room temperature. The chip was washed 3 times in PBST and 3 times inPBS and 1×10¹² phage prepared from the human non-immunised library werethen incubated on the chip surface for 1 hour at room temperature. Thechip was washed as before and 100 μl of biotin tyramine mix (asdescribed in Example 2 part bi) was incubated on the chip for 10 min atroom temperature. The chip was washed as before and phage eluted fromthe chip using 200 μl of triethylaime TEA. The TEA was neutralised with100 μl of 1M Tris-HCl pH 7.4.

ii) Recovery of biotinylated phage

20 μl of streptavidin-coated magnetic beads (Dynal) were taken out ofsolution using a magnet and blocked for 2 hours at room temperature on arotating platform with 1 ml of 3% MPBS. Beads were pelleted and 300 μlof eluted phage with 60 μl of 15% MPBS were added to the blocked beadsand rotated for 15 minutes at room temperature. Beads were pelleted,washed three times in 1 ml PBST and three times in 1 ml PBS. The beadswere resuspended in a final volume of 100 μl PBS. 50 μl of this wastaken and used to directly infect 5 ml of an exponentially growingculture of E coli TG1 at 37° C. for 1 hour with light aeration in 2TYAGmedium. Infected TG1s were plated on 2TYAG medium in 243 mm×243 mmdished (Nunc). Dilutions of bacteria were also plated out and incubatedat 30° C. overnight. Colony counts gave the phage output titre.

iii) Second round of selection.

Colonies were scraped off the 243 mm×243 mm plates into 3 ml of 2TYbroth and 15% (v/v) glycerol added for storage at −70° C. Glycerol stocksolutions from the first round of selection of the repertoire on theTGFβ1 BIACORE® (biosensor instrument, Pharmacia) chip were rescued usinghelper phage to derive phagemid particles for the second round ofselection. 250 μl of glycerol stock was used to inoculate 50 ml 2YTAGbroth, and incubated in a 250 mL conical flask at 37° C. with goodaeration until the OD600 nM reached 0.7 (approximately 2 hours). M13K07helper phage (moi=10) was added to the culture which was then incubatedstationary at 37° C. for 15 minutes followed by 45 minutes with lightaeration (200 rpm) at the same temperature. The culture was centrifugedand the supernatant drained from the cell pellet. The cells wereresuspended in 50 ml prewarmed 2YTAK, and the culture incubatedovernight at 30° C. with good aeration. Phage particles were purifiedand concentrated by PEG precipitation (Sambrook et al., 1990) andresuspended in PBS to 10¹³ tu/ml.

The second round of selection and capture of biotinylated phage on theTGFβ1-BIACORE® (biosensor instrument, Pharmacia) chip was carried outexactly as the first round. The phage output titres are shown below.

Strepavidin captured- Total output output % captured Round 1 2.5 × 10⁷   5 × 10⁵ 2 Round 2   6 × 10¹⁰ 1.8 × 10⁵ 0.5

d. Growth of single selected clones for soluble ELISA to identifyanti-TGFβ1 scFv

Single colonies were grown up exactly as described in Example 2 part cand the ELISAs were carried out as in part d, except that the plateswere coated with 0.2 μg/ml recombinant TGFβ1 (R and D Systems). The 192clones from the second round of selection were screened by ELISA and 26were found to be TGFβ1 positive (13.5%).

e. Sequencing of anti-TGFβ1scFv Antibodies

The nucleotide sequences of the anti-TGFβ1 antibodies were determined byfirst using vector-specific primers to amplify the inserted DNA fromeach clone. Cells from an individual colony on a 2YTAG agar plate wereused as the template for a polymerase chain reaction (PCR) amplificationof the inserted DNA using the primers pUC19reverse and fctetseq.Amplification conditions consisted of 30 cycles of 94° C. for 1 min, 55°C. for 1 min and 72° C. for 2 min, followed by 10 min at 72° C. The PCRproducts were purified using a PCR Clean-up Kit (Promega) in to a finalvolume of 50 μl H₂O. Between 2 and 5 μl of each insert preparation wasused as the template for sequencing using the Tag Dye-terminator cyclesequencing system (Applied Biosystems). The primers mycseq10 andPCR-L-Link were used to sequence the light chain of each clone andPCR-H-Link and pUC19reverse to sequence the heavy chain.

Sequencing revealed that a total of six different arti-TGFβ1 antibodieshad been isolated by the signal transfer selection method using the31G9-HRP conjugate to target the site specific biotinylation. These sixantibodies were of VH germlines different from that of 31G9, as shownbelow.

CLONE VH GERMLINE VL GERMLINE ST3 VH3 DP53 VLambda2 DPL11 ST6 VH3 DP53VLambda3 DPL16 ST10 VH3 DP53 VLambda3 DPL16 ST14 VH3 DP53 VLambda2 DPL12ST19 VH3 DP53 VKappa1 DPK9 ST21 VH3 DP53 VLambda2 DPL12 31G9 VH3 DP49VKappa1 DPK9

All the clones selected had the same VH3 DP53 germline paired with avariety of VL gene segments. ST6 and ST10 had the same VL germline anddiffered from each other at a single amino acid residue in VL CDR2. ST14and ST21 also had the same VL germline but differed from each other at asingle amino acid residue in VL CDR3. None of the selected clones hadthe same VH as 31G9. Clone ST19 had the same germline VL as 31G9 with asingle amino acid change in VL FR2.

Overall this demonstrates the ability of the signal tansfer selectiontechnique to select away from an undesired antigenic epitope andgenerate new lineages of phage antibodies which may have alteredspecificities.

EXAMPLE 9 Selection of Anti-Chemokine Receptor Phage using a ChemokineLigand to Guide Selection

The chemokine receptor CC-CKR5 is a co-receptor for macophage tropicHIV-1 strains which is expressed on CD4⁺ lymhocytes. CC-CKR-5 respondsto a number of chemokines, including macrophage inflammatory protein(MIP)-1α. MIP-1α also binds to other chemokine receptors, includingCC-CKR1 and CC-CKR4. MIP-1α may be used to guide signal transferselection of phage antibodies or other phage displayed proteins whichbind to the CC-CKR5 receptor.

a. Preparation of human CD4+ cells from blood.

Mononuclear cells were prepared from a 50 ml buffy coat usingFICOLL-PAQUE®, (Aqueous solution of a nonionic synthetic polymer ofsucrose density optimized for lymphocyte isolation from whole blood,Pharmacia) density gradient centrifugation (600 g for 20 min at 20° C.).CD4⁺ cells were then isolated from the 1.5×10⁸ recovered cells using aBiotex CD4 column, following the manufacturer's instructions, althoughPBS/2% foetal calf serum (FCS) was used throughout. Eluted cells werepelleted at 600 g for 5 min and resuspended in 300 μl PBS/2% FCS.8.3×10⁶ cells were recovered using this procedure. The recovered cellswere analysed by flow cytometry and approximately 59% of the cells werefound to be CD4⁺.

b. Selection procedure and capture of biotinylated phage.

1×10⁵ CD4⁺ lymphocytes were incubated with 2×10¹² phage prepared fromthe 1.4×10¹⁰ scFv phage display library in either the presence orabsence of biotinylated MIP-1α (R and D Systems) at a finalconcentration of 375 nM. The final volume for each selection was made up40 μl with PBS containing 2% marvel (MPBS). Selections were incubatedfor 14 hr at 4° C. Cells were pelleted by centrifugation at 600 g for 3min, and washed in 1 ml MPBS. A total of three washes were carried out.100 μl of streplavidin-HRP was added at a dilution of 1:1000 in MPBS.This was incubated for 2 hr, then washed as before. Biotin tyramine wasthen added (as Example 2, part bi) in 100 μl of 150 mM NaCl/50 mMTrisHCl pH 7.4 containing 3% H₂O₂ and incubated for 10 min at roomtemperature. Cells were washed and resuspended in 100 μl TE containing0.5% triton. Biotinylated phage were captured on 10 μl of MPBS-blockedstreptavidin-coated magnetic beads (Dynal). The beads were washed threetimes in 1 ml PBS/0.1% Tween 20 (PBST), then resuspended in 100 μl ofPBS. Phage eluate before and after streptavidin capture were titered byinfection of an exponentially growing culture of E coli TG1 at 37° C.for 1 hr. The numbers of phage recovered from the various selectionprocedures are shown below.

Strep-HRP Selection Bio- No. phage Total No. % phage No. MIP-1αBio-tyramine Eluted Captured Captured 1 + + 3.7 × 10⁵ 5.9 × 10³ 1.6 2 +− 4.0 × 10⁵ 8.0 × 10² 0.2 3 − + 4.9 × 10⁵ 1.4 × 10³ 0.3

The greatest recovery of biotinylated phage was observed from CD4⁺lymphocytes incubated with both the biotinylated MIP-1α and biotintyramine. Omission of either the biotinylated ligand or the biotintyramine resulted in an approximately 5 to 6-fold drop in the percentageof phage recovered from the eluate. These results suggest thebiotinylated MIP-1α is capable of binding the CD4⁺ cells in the presenceof the phage library and directing biotinylation of phage binding aroundit in the presence of HRP and hydrogen peroxide.

c. Phage ELISA to identify CD4⁺ cell binders, CC-CKR5 transfected cellbinders and CC-CKR5 amino terminal peptide binders.

Selected phage were analysed by phage ELISA for their ability torecognise CD4⁺ lymphocytes, a CC-CKR5 transfected cell line (provided byM. Parameter and G. Vassart, University of Brussels) and aBSA-conjugated peptide corresponding to the amino terminal twenty aminoacids of the CC-CKR5 receptor (MDYQVSSPIYDINYYTSEPC SEQ ID NO:32). PhageELISAs were carried out as follows: individual clones were picked into a96 well tissue culture plate containing 100 μl 2YTAG. Plates wereincubated at 37° C. for 6 hours. M13KO7 helper phage was added to eachwell to an moi of 10 and incubated with gentle shaking for 45 min at 37°C. The plates were centrifuged at 2000 rpm for 10 min and thesupernatant removed. Cell pellets were resuspended in 100 μl 2TYA withkanamycin (50 μg/ml) and incubated at 30° C. overnight. The ELISA wasthen carried out as for soluble ELISA (Example 2) except that in placeof the 9E10 a goat anti-M13 antibody was used at a dilution of 1:2500,followed by an anti-goat alkaline phosphatase conjugate, also at adilution of 1:2500. 1×10⁵ cells per ELISA well were used and the peptideBSA conjugate was coated at a concentration of 1 μg/ml.

30/95 of the phage selected in the presence of biotin tyramine andMIP-1α recognised CD4⁺ lymphocytes. 11/95 of the phage selected in theabsence of MIP-1α recognised CD4⁺ lymphocytes. 13 of the 30 clones whichwere positive on CD4⁺ cells were also found to be positive on theCC-CKR5 cell line. Of these two clones (RK-1 and RK-2) selected in thepresence of MIP-1α and biotin tyramine were found to be specific for theCC-CKR5 peptide. The clones which do not recognise the CC-CKR5 peptidemay of course recognise other epitopes of CC-CKR5, other MIP-1αreceptors or proteins which are found on the cell surface in closeproximity to MIP-1α receptors.

d. Sequencing of RK1 and RK2.

Sequencing of the two peptide binding clones was carried out asdescribed in Example 2 part f. Clones RK1 and RK2 had identical VL genesegments.

VH VH VL VL family segment family segment RK1 VH4 DP67 Vl3 DPL16 RK2 VH4DP14 Vl3 DPL16

e. Western blotting using RK2

A representative of these peptide-binding clones (RK-2) was tested bywestern blotting on extracts from a CC-CKR5 transfected cell line andwas found to bind to an approximately 35 kD band which may correspond toCC-CKR5.

This work describes the use of signal transfer selection isolate phageantibodies of a desired specificity directly from a large phage libraryusing a ligand of a known binding specificity (MIP-1α) as a marker toguide selection of phage binding in an area around the ligand bindingsite. A proportion of the resultant selected population has been shownto be specific for the ligand's receptor (CC-CKR5). The antibodiesgenerated in this example bind to a seven transmembrane protein whichacts as a co-factor in HIV infection, hence the antibodies may have atherapeutic role.

EXAMPLE 10 Selection of Anti-Chemokine Receptor Phage usingLight-Activated Streptavidin and the Receptor Ligand to Guide

As described in Example 9 MIP-1α can be used to guide selection ofantibodies to at least one of its receptors (CC-CRK5). This exampleutilises the same system to demonstrate to ability of light activatiblestreptavidin to be used instead of biotin tyramine in an analogoussignal transfer procedure.

a. Generation of light activatible streptavidin.

SAND (sulphosuccinimidyl2-[m-azido-o-nitrobenzamido]-ethyl-1,3-dithiopropionate, Pierce) is aphotocrosslinking agent which is activated in the visible range (300-460nm). SAND was linked to streptavidin by mixing 2 mg/ml streptavidin(Pierce) 7.5 mM SAND in PBS. This was incubated in the dark room at roomtemperature for 2 hr. then separated on a NAPS column.

b. Selection procedure

1×10⁵ CD4⁺ lymphocytes were prepared as described in Example 9 part aand incubated with 2×10¹² phage prepared from the 1.4×10¹⁰ scFv phagedisplay library in either the presence or absence of biotinylated MIP-1α(R and D Systems) at a final concentration of 375 nM. The final volumefor each selection was made up 40 μl with PBS containing 2% marvel(MPBS). Selections were incubated for 14 hr at 4° C. Cells were pelletedby centrifugation at 600 g for 3 min, and washed in 1 ml MPBS. A totalof three washes were carried out. Cells were then incubated in the darkfor 30 min with 500 mM streptavidin-conjugated SAND. Cells were washedas before, the exposed to 5 flashes of light from a standard flashgun.Cells were pelleted and resuspended in 100 μl TE containing 0.5% triton.

c. Captured of streptavidin-linked phage

The eluate was added to preblocked immunosorb tubed coated with 1 ml of100 μg/ml biotinylated-BSA. After 1 hour the tube was washed 10 times in1 ml PBS. Phage which had been cross-linked to the streptavidin wereeluted in 1 ml PBS containing 28 mM b-mercaptoethanol. Phage from thetotal eluate and from the captured population were titred. The numbersof phage recovered from the various selection procedures are shownbelow.

Selection Strep Total No. No. phage % phage No. Bio-MIP-1α SAND ElutedCaptured Captured 1 + + 8.2 × 10⁴ 54 0.06 2 + − 3.6 × 10³ 0 0 3 − + 4.4× 10⁵ 0 0

Phage were only recovered from the final eluate when streptavidin-SANDwas included in the selection scheme. In the absence of this nobackground phage were recovered. These results deomstrate the ability ofbiotinylated MIP-1α and a light activatible streptavidin molecule tospecifically cross-link streptavidin to phage binding around the site ofMIP-1α binding.

c. Phage ELISA to identify CD4⁺ cell binders, CC-CKR5 transfected cellbinders and CC-CKR5 amino terminal peptide binders.

Selected phage were analysed by phage ELISA for their ability torecognise CD4⁺ lymphocytes, a CC-CKR5 transfected cell line (provided byM. Paramentier and G. Vassart, University of Brussels) and aBSA-conjugated peptide corresponding to the amino terminal twenty aminoacids of the CC-CKR5 receptor (MDYQVSSPIYDINYYTSEPC SEQ ID NO:32). PhageELISAs were carried out as described in Example 9.

24/54 of the phage selected in the presence of biotin tyramine andMIP-1α recognised CD4⁺ lymphocytes. 15 of the 24 clones which werepositive on CD4⁺ cells were also found to be positive on the CC-CKR5cell line. Of these two clones (RK-3 and RK-4) were found to be specificfor the CC-CKR5 peptide.

d. Sequencing of RK3 and RK4.

Sequencing of the two peptide binding clones was carried out asdescribed in Example 2 part f.

VH VH VL VL family segment family segment RK3 VH4 DP14 Vλ3 DPL16 RK4 VH4DP14 Vλ3 DPL16

RK1, which was a clone generate by biotin tyramine signal transferselection using MIP-1α as a guide molecule was identical to RK-3, withthe exception of a single amino acid difference in the VL CDR3. Thisdemonstrates the selectivity of the selection procedures; a virtuallyidentical clone recognising the same CC-CKR5 region can be selected byeither biotin tyramine or light activatible-streptavidin signal transferselection from a background of 1.4×10¹⁰ other clones.

EXAMPLE 11 Selection of Phage Antibodies to Two Different Cell SurfaceAdhesion Molecules using a Biotinylated Ligand Which Binds to Both toGuide Selection

E and P selectin are cell adhesion molecules which are expressed on thesurface of human vascular endothelial cells (HUVECs). E and P selectinare upregulated after stimulation with thrombogenic or inflammatoryagents such as TNFα. The ligand for both these selectin has been foundto be sialyl Lewis X, and this ligand has been used to generateantibodies to both of its receptor adhesion molecules in the sameselection.

a. Stimulation of HUVEC's using TNFα.

HUVEC's (grown to passage 5) were stimulated with TNFα at 500 pg/ml for4 hours and flow cytometry analysis was carried out to ensure that Eselectin was up-regulated. After stimulation 43.8 percent of the cellstreated gave a fluorescence value greater than 1, whereas withoutstimulation only 2.6 percent of the cells gave fluorescence greater than1.

b. Biotinylation of sialyl Lewis X.

Sialyl Lewis X (Oxford Glycosystems) was biotinylated using biotinylateddiaminopyridine (BAP). 1 mg BAP was dissolved in 50 μl pyridine/aceticacid (2:1 v/v). This was added directly to the dry carbohydrate (100 μg)and incubated for 1 hour at 80° C. The oligosaccharide-BAP adducts werereduced by the addition of 50 μl of 2.1M/l borane dimethylamine inpyridine/acetic acid and vortexed. Incubation was then carried on for afurther hour at 80° C.

c. Selection and capture of biotinylated phage.

Stimulated HUVEC's were incubated with phage rescued from the largenon-immunised scFv library. Two rounds of signal transfer selectionswere carried out in the presence or absence of 40 μg of biotinylatedsialyl Lewis X. Phage were captured on streptavidin-coated magneticbeads as described in Example 1 part (e). The number of phage presentbefore and after capture was titred. The greatest recovery ofbiotinylated phage was observed from stimulated cells when biotinylatedsialyl Lewis X and biotin tyramine steps were present (1.8% recovery).Omission of either the biotinylated sialyl Lewis X or biotin tyramineresulted in an approximately 10-fold drop in the % of phage recoveredfrom the eluate (both gave 0.2% recovery). These results suggest thatbiotinylated sialyl-Lewis X (with streptavidin-HRP) is capable ofbinding to the stimulated HUVEC's in the presence of the phage libraryand directing biotinylation of phage binding around the ligand bindingsites.

d. Soluble ELISA to identify E- and P-selectin binders.

Recovered phage were examined by soluble ELISAs [as described in Example2 part(d)] for their binding to E and P selectin. 3.6% of the clonesrecovered from the first round of selection in the presence ofbiotinylated sialyl Lewis X and biotin tyramine were E selectinpositive. None of the clones tested from selections carried out onunstimulated cells, or in the absence of ligand or biotin tyramine wereE selectin positive. 2.8% of the clones recovered from theHRP-conjugated anti-E selectin IgG selections were found to bind Eselectin, whereas in the absence of the HRP-conjugate no clones werefound to be E selectin positive. From the second round of selection inthe presence of sialyl Lewis X and biotin tyramine the number of clonesfound to be E selectin positive increased to 13.7%.

P selectin ELISAs were also carried out on the population of clonesselected in the presence of biotinylated sialyl Lewis X and biotintyramine on stimulated cells 50% of the E selectin binders were alsofound to recognised P selectin, which shares sialyl Lewis X as itsligand. In addition a further 2% were found to be P selectin specific.

A further 21% of the second round selected population were found to bindto stimulated HUVEC's by soluble ELISA. The clones found to bind Eselectin were sequenced and a diverse population of E selectin binderswere identified. A range of different germline VH's were selected. TheVL's were less diverse; a total of 4 different germline segments wereselected which had common CDR3's.

These selections demonstrate the ability of a natural ligand for aparticular cell surface protein to direct selection of cell surfaceprotein binding clones. A ligand which recognises more than one cellsurface protein (in this case E and P selectin) can be used to guideselection of antibodies to either of its target proteins.

EXAMPLE 12 Measurement of the Distance over which Signal Transfer usingBiotin Tyramine May Occur

The following experiment was designed to assess the distance over whichbiotinylation may occur using HRP and biotin tyramine. Bacteriophage areapproximately 1 μm long filaments with three copies of the gene 3protein at one end of the filament. The gene 3 protein provides a markerwhich can be used to localise HRP specifically to one end of the phagevia a mouse anti-gene 3 antibody, followed by an anti-mouse-HRPconjugate. Biotin tyramine and hydrogen peroxide can then be added tothe tagged phage to allow biotinylation of the phage around the site ofthe HRP activity. Phage can then be transferred to electron microscopegrids and labelled with streptavidin-gold beads to visualise the extentof biotinylation.

a. Preparation of phage.

An oestradiol-binding phage (MT31C) was grown from a bacterial glycerolstock in 50 ml 2TY/2% glucose/1 μg/ml ampicillin (2TYGA) for 6 hours at37° C. M13KO7 helper phage (Stratagene) was added to the culture to amultiplicity of infection (moi) of approximately 10 (assuming that an OD600 mm of 1 is equivalent to 5×10⁸ cells per ml of culture). The culturewas incubated stationary at 37° C. for 15 minutes followed by 45 minuteswith light aeration (200 rpm) at the same temperature. The culture wascentrifuged and the supernatant drained from the cell pellet. The cellswere resuspended in 50 ml 2TYAK (2TY media supplemented with 100 μg/mlampicillin and 50 μg/ml kanamycin), and the culture incubated overnightat 30° C. with good aeration (300 rpm). Phage particles were purifiedand concentrated by two polyethylene glycol (PEG) precipitations(Sambrook, J., Fritsch, E. F., and Maniatis, T. (1990). Molecularcloning—A Laboratory Manual. Cold Spring Harbour, N.Y.) and resuspendedin PBS to 10¹² transducing units (tu)/ml.

b. Biotinylation of phage.

Phage were diluted to an approximate concentration of 2×10¹⁰ per ml in atotal volume of 500 μl. 5 μl of mouse Mab directed agains the gene 3protein were then added to the phage and incubated at room temperaturefor 1 hour. 5 ml of an anti-mouse-IgG-HRP conjugate (Sigma) were thenadded to the phage and incubated at room temperature for a further 1hour. The phage were then treated with biotin tyramine by adding 50 μlof 1M Tris-HCl pH 7.4 to the phage mix, followed by 4 μl of biotintyramine stock solution and 2 μl of hydrogen peroxide (Sigma). Thereaction was allowed to proceed at room temperature for 10 minutes, andthe biotinylated phage then stored at 4° C. overnight.

c. Streptavidin-gold labelling of biotinylated phage.

EM grids were blocked in 0.1% gelatin and phage samples then applied.The phage were then labelled with streptavidin-5 nm colloidal gold(Sigma) at an approximate concentration of 2×10¹¹ particles per ml. Anumber of images of the biotinylated ends of phages were generated. Whenthe anti-gene 3 antibody was omitted no gold labelling of the phage endscould be observed.

d. Estimation of the distance over which biotinylation has occurredusing this system.

The number of gold particles found to localise to individual ends ofphages in the electron micrographs were counted and the data were usedto generate a distribution histogram (FIG. 2). Data from two separatelabelling experiments were pooled to generate the histogram. The averagenumber of gold particles associated with the phage ends was found to be6.6, giving an average radius of biotinylation of 7.2 nm. Using thismethod the biotinylation range observed was from 5 nm to 25 nm, 5 nmbeing the limit of resolution of the experiment. A typical globularprotein has a diameter of around 4 nm, hence the biotinylation range isof the order of 1 to 5 protein diameters.

e. Adjusting the distance of biotinylation.

To increase the distance over which biotinylation occurs HRP-conjugatedmolecules of various lengths may be used. For example, a phage antibodywith a specific binding characteristic may be HRP labelled and then usedto guide the biotinylation of phage antibodies from the library. A phageparticle is normally around 1 μm long, hence this would give a radius ofbiotinylation of 10 nm to 1 μm. Similarly other molecules of shorter orlonger lengths may be used e.g. streptavidin-dextran-HRP conjugates, orbeads of defined sizes such as MACS® beads (magnetic cell sorting beads,(Miltenyi Biotec) which have a diameter of 50 nm and can be coupledeither directly, or indirectly via biotin-streptavidin, to HRP.Iterations of the biotin tyramine reaction may be performed to broadenthe area over which biotinylation is occurring. Example 10 describes avariation on the signal transfer technique which uses a lightactivatible streptavidin molecule with a short spacer arm (18 Angstrom).This procedure will only allow signal transfer to molecules bindingimmediately adjacent to the guide molecule.

EXAMPLE 13 Step-Back Selection to Isolate Phage Antibodies which InhibitLigand Binding

This example describes use of the biotin tyramine signal transferselection procedure in a two step manner to isolate antibodies whichinhibit binding of the initial marker ligand to cells. This proceduremay be applied to the generation of inhibitors to any ligand, smallmolecule, or antibody. The process as exemplified here involves aninitial first stage of the selection to biotinylate and capture phageantibodies which bind around the site of ligand binding. Thebiotinylared phage are then used directly (with no need foramplification) to guide a second stage of selection using cells in theabsence of ligand. In this way antibodies which bind in the ligandbinding site can be biotinylated by signal transfer procedure, thencaptured and screened for inhibition of ligand binding. Such a scheme isoutlined in FIG. 3. The example described here uses phage to direct thesecond stage selection, but scFv may also be used either as a populationof scFv molecules, or by individual clone isolation and purification, asmay any other suitable binding molecule such as an antibody or bindingfragments thereof. The system used in this example is the same as thatdescribed in Example 9. MIP-1α was used as the guide ligand on purifiedCD4+ lymphocytes.

a. First stage selection

CD4+ cells were purified from blood as described in Example 9 part a.The first stage selection procedure was then carried out exactly asdescribed in Example 9 part b), except that phage were captured on 90 μlpreblocked streptavidin-coated Dynal beads. After washing the beads wereresuspended in 90 μl PBS and 30 μl removed to titre the phage present onthe beads. The remaining 60 μl were again taken out of solution usingthe magnet and phage were eluted from the beads using 100 μl l 100 mMtriethlyamine for 10 minutes at 37° C., and then neutralised with 50 μl1M Tris-HCl pH 7.4. After elution the beads were taken out of solutionand the supernatant containing the biotinylated phage was taken for usein seep two. The remaining beads were retained and used to infect E coliTG1 to ascertain the phage titre remaining on the beads after elution.

The following titres were obtained:

Total number of phage captured on the Dynal beads: 1.7×10⁴

Total number of phage retained an the beads after TEA elution: 2.2×10³

Therefore total number eluted: 1.5×10⁴

b. Second stage selection

The population of biotinylated phage which had been recovered from thefirst stage of the selection was added directly to 1×10⁶ CD4+lymphocytes in a total volume of 200 μl in MPBS. Phage were allowed tobind to the cells for 1 hr at room temperature, and cells were thenwashed 3 times in 1 ml PBS. Cells were pelleted at 4000 rpm for 2 min ina minifuge between washes. A further aliquot of the scFv phage library(2×10¹² phage) was then added to the cells in 1 ml MPBS and allowed tobind for 1 hr at room temperature. Cells were washed 3 times in PBS asabove and then resuspended in 200 μl MPBS containing 2 μl ofstreptavidin-HRP complex (Amersham). This was allowed to bind for 30 minat room temperature, and cells were then washed as before. Biotintyramine treatment of the cells was carried out as described in Example9 part b). Cells were then lysed by resuspension in 100 μl PBST and 30μl preblocked streptavidin-coated Dynal beads were added to the lysate.Beads and lysate were rotated at room temperature for 20 min, and thebeads then taken out of solution on a magnet. Beads were washed 3 timesin 1 ml PBST, followed by 3 times in 1 ml PBS. Washed beads were used todirectly infect an exponentially growing culture of E coli TG1. A totalof around 4×10³ clones were recovered from this selection procedure.

c. Growth of single selected clones for immunoassay.

Individual colonies from the second step of the selection procedure wereused to inoculate 100 μl of 2TYGA into individual wells of tissueculture plates. Plates were incubated at 30° C. overnight with moderateshaking (200 rpm). Glycerol to 15% was added to each well and thesemaster plates stored at −70° C. until ready for analysis.

d. Phage ELISA to identify anti-CD4+ scFv's.

Cells from the master plate were used to inoculate fresh 96 well tissueculture plates containing 100 μl 2TYGA per well. These plates wereincubated at 37° C. for 6-8 hr. M13KO7 was added to each well to an moiof 10 and incubated stationary for 30 min then 30 min with gentleshaking (100 rpm), both at 37° C. The plates were centrifuged at 2000rpm for 10 min and the supernatant removed. Each cell pellet wasresuspended in 100 μl 2TYAK and incubated at 30° C. overnight. Eachplate was centrifuged at 2000 rpm for 10 min and the 100 μl ofsupernatant was recovered and blocked in 20 μl 18% M6PBS (18% skimmedmilk powder, 6×PBS), stationary at room temperature for 1 hr.

CD4+ cells were isolated as described (Example 9, part a) and 1×10⁵cells were spun onto 96 well culture wells which had been precoated withpoly-L-lysine for 30 min at room temperature. Cells were blocked in 100μl MPBS for 2 hours at 37° C., and rinsed once in PBS. The phagesupernatants were then added to the cells and incubated for 1 hr at roomtemperature, then washed 3 times in PBS. 100 μl of a 1:5000 dilution ofsheep anti-fd antibody (Pharmacia) in MPBS was added and the platesincubated at room temperature for 1 hr. Plates were washed 3 times inPBS and 100 μl of a 1:5000 dilution of donkey anti-sheep alkalinephosphatase conjugate (sigma) in MPBS was added and incubated for 1 hrat room temperature. Plates were washed 3 times in PBS and alkalinephosphatase activity was visualised using the chromagenic substrate pNPP(Sigma). Absorbance was measured at 405 nm using a microtitre platereader. 45 individual colonies were assessed for CD4 cell binding inthis way, and 25 were found to be positive. 6 of these were taken atrandom for further analysis.

e. Assessment of anti-CD4 scFv's to inhibit binding of MIP-1α to CD4+cells.

6 scFv's were purified using nickel agarose metal affinitychromatography (Quiagen). 1×10⁵ CD4 cells were preincubated with thepurified CD4+-binding scFv's, or with an irrelevant control scFv for 1hr at room temperature in PBS containing 0.1% BSA in a total volume of100 μl. Approximately 5-10 μg of scFv was used per sample. Cells werepelleted at 4000 rpm in a minifuge and washed once in 1 ml PBS.Biotinylated MIP-1α (R and D Systems) was made up according tomanufacture's instructions 5 μl (equivalent to 5 ng) added to the cellsin 100 μl MPBS and incubated at room temperature for 1 hr. Cells werewashed as before. 100 μl of streptavidin-FITC (Sigma) at a dilution of1:100 in MPBS was added and incubated for 30 min at room temperature,and cells were washed as before. Fluorescence was detected using anEPICS-XL® flow cytometer (Coulter Cytometry, Miami, Fla.). MIP-1α gavesignificant shift in the fluorescence of the cells when no scFv, orcontrol scFv was added to the cells. In the presence of scFv from theselected clones MIP-1α binding to the cells was significantly inhibited.Inhibition varied from clone to clone.

EXAMPLE 14 Biotin Tyramine Selection in Solution using a Peptide PhageLibrary

9E10 is a commercially available mouse monoclonal antibody whichrecognises aeptide which is part of the Cellular myc protein (Munro, S.and Pelham, H. R. B. (1986), Cell 46, 291-300). This experiment wasdesigned to select for peptides from a large peptide library which bind9E10. 9E10 was conjugated to HRP to allow biotin tyramine-directedselection in solution. This can be considered as a novel method ofepitope mapping antibodies, or other protein binding domains.

a. Construction of the peptide library

In this example, the peptide library used as constructed as described byFisch et al (I. Fisch et al (1996) Proc. Natl. Acad. Sci. USA 937711-7766) to give a phage display library of 1×10¹³ independent clones.

b. Conjugation of the anti-myc antibody (9E10) to HRP

1 ml of 1 mg/ml 9E10 IgG was conjugated to HRP using the Pierce EZ-linkmalemide activated HRP kit (cat no. 31494). 1 ml of 9E10 was added tothe vial containing 6 mg of 2-mercaptoethylamine (MEA) in 100 μlconjugation buffer. This was incubated for 90 min at 37° C. The solutionwas allowed to cool to room temperature and the MEA was separated fromthe reduced IgG using the desalting column. The column waspre-equilibrated by washing with 30 ml of maleimide conjugation bufferand the 1.1 ml of IgG/MEA solution was applied to the column. Theconjugate was eluted using the maleimide conjugation buffer. 1 mlfractions were collected and fractions 5 and 6 were found to contain themajority of the protein. Fractions 7 and 8 contained smaller amounts ofprotein and were retained for control selections. Fractions 5 and 6 werepooled and 1 mg of maleimide activated HRP was added to the IgG andallowed to react for 1 hour at room temperature.

c. First round selections using 9E10-HRP and the peptide phage displaylibrary

Approximately 1×10¹³ phage were used per selection. 6 μg of 9E10-HRPconjugate were added to the peptide phage in a total volume of 1 ml PBSwith 2% marvel (MPBS). Control selections were also carried out using 6μg of unconjugated 9E10. All selections were carried out in 1 ml. Phageand antibody were allowed to bind overnight at 4° C. 50 μl 1M Tris-HClpH 7.4, 4 μl biotin tyramine and 2 μl hydrogen peroxide were then addedto the selection and allowed to react for 10 min at room temperature.100 μl of streptavidin-coated magnetic beads (Dynal) which had beenpreblocked for 30 min in MPBS were then added to the selection androtated at room temperature for 30 min. Magnetic beads were then broughtout of solution using a magnet and washed with 3×1 ml of PBS containing0.1% Tween, followed by washing with 3×1 ml of PBS. The beads wereresuspended in a final volume of 100 μl PBS and used to directly infect5 ml of an exponentially growing culture of E. coli TG1. Infection wascarried out by incubation stationary at 37° C. for 30 min, followed by30 min slow shaking (200 rpm) at 37° C. Phage were plated out on 2TYmedium containing 100 μg/ml tetracyclin (2TYT). Colony counts gave thephage titre.

d. Second and third round selections using 9E10-HRP and the peptidelibrary.

The plates were scraped into 5 ml of 2TY. 50 μl of this plate scrape wasthen added to 50 ml of 2TYT and grown overnight at 30° C. with aeration1 ml of the resultant cell suspension was pelleted at 6000 rpm in aminifuge and 100 μl of 10×MPBS added to the supernatant. 9E10-HRPconjugate, unconjugated were then added to the blocked phage asselections carried out exactly as the first round described in part c.above. The selection was repeated so that a total of three rounds ofselection were performed. The number of phage recovered in the outputpopulations at each round was as follows:

9E10-HRP 9E10 Round 1 5.2 × 10⁵ 2.0 × 10⁴ Round 2 1.2 × 10⁶ 5.9 × 10⁴Round 3 8.0 × 10⁵ 2.6 × 10⁵

e. Screening the output populations for binding to 9E10

Individual colonies from the third round of selection were used toinoculate 96 well tissue culture plates containing 100 μl of 2TYT perwell and clones were grown overnight at 30° C. with good aeration (300rpm). Plates were centrifuged at 2000 rpm and the 100 μl fromsupernatant from each well was recovered and blocked in 20 μl 18% M6PBS(18% milk powder, 6×PBS) stationary at room temperature for 1 hour.ELISA plates which had been blocked overnight at 4° C. with 50 μl of 10μg/ml 9E10, or 50 μl PBS alone were washed in PBS and then blocked for 2hours stationary at 37° C. in 3MPBS. ELISA plates were washed in PBS andthe blocked phage supernatants then added to the ELISA plate. The plateswere incubated stationary at room temperature, then washed three timeswith PBST, followed by three washes with PBS. 50 μl of anti-gene 8-HRPconjugate diluted at 1:5000 in 3MPBS were then added to each well andthe plates incubate at room temperature for 1 hour. Plates were washedas before and the ELISA developed for 1 hour at room temperature with 50μl of TMB substrate. Development was stopped by the addition of 25 μl of1M H₂SO₄.

f. Results of the screening

95 clones from the third round of selection using the 9E10-HRPconjugate, and 95 from the unconjugated 9E10 selection were screened byELISA. 3 positives were identified as binding 9E10, but not PBS coatedplates from the 9E10-HRP selection, whereas no positives were found fromthe control unconjugated 9E10 selection. The three positive clones wererechecked by ELISA as above on an unrelated mouse monoclonal antibodyand did not give any signal, demonstrating that they bind specificallyto the 9E10 Mab.

g. Sequencing 9E10-binding clones

Clones found to be positive for binding to 9E10 were analysed by DNAsequencing as described by Fisch et al. All three clones were found tobe identical. None had a peptide insert in Exon 1, and all a 10 aminoacid peptide sequence inserted in Exon 2 which had some homology to themyc tag, as shown below:

Selected sequence SEQ ID NO33: P M P H A E G K S T

Myc tag SEQ ID NO34: G A A E Q K L I S E E D L M

In summary 9E10-specific clones have been identified from the peptidelibrary, which have some homology to the myc tag. This demonstrates thatbiotin tyramine selections can be successfully carried out in solution,and can be carried out on non-antibody libraries.

EXAMPLE 15 Characterisation of Clones Which Bind to CD4+ Cells, but notto the Chemokine Receptor CC-CKR5, by Western Blotting and ICC

Example 9 described the selection of phage antibodies which bind to achemokine receptor. Phage selections were carried out on CD4+ cellsusing biotinylated MIP-1α, followed by streptavidin-HRP to guide theselection. 30/95 phage selected in the presence of the biotin tyramineand MIP-1α recognised CD4+ lymphocytes. 13 of these clones were found tobe positive for the CC-CKR5 chemokine receptor for which MIP-1α is aligand, leaving 17 clones which bind to CD4+ cells, but to anotherantigen to be discertained (Example 9 part c). These clones mayrecognise antigens which are normally found in close proximity to MIP-1αreceptors, or are MIP-1α receptors other than CC-CKR5 (CC-CKR1 andCC-CKR4 both bind to MIP-1α). Identification of the antigens to some ofthese CD4+-binding clones allows examination of protein-proteininteractions on the cell surface, and exemplifies the potential ofbiatin tyramine selection as a tool for discovering novelprotein-protein interactions.

Three clones, CD4A2, CD4E1 and CD4D2 were chosen at random from the 17CD4+ binding clones and were subjected to further analysis to identifytheir antigen partners. Initial studies involved probing western blotsof membrane fractions prepared from CD4+ cells with purified scFv fromthe 3 clones. Immunocytochemistry on CD4+ cells was also carried outusing the scFv's.

a. Preparation of CD4+ cell membrane fractions.

CD4+ lymphocytes were prepared as described in Example 9, part (a).Membrane preparations were then generated as follows. Approximately1×10⁶ cells were resuspended in 1 ml of 12 mM Tris-HCl, pH 7.5 in 250 mMsucrose. Cells were lysed by three cycles of freeze thawing, and thelysates were homogenised in a ground glass homogeniser. The homogenatewas centrifuged at 270×g for 10 min at 4° C. to pellet the nuclearfraction. The supernatant was then centrifuged at 8000×g for 10 min at4° C. to pellet the mitochondrial and lysosomal fractions. The finalcentrifugation to pellet the plasma membrane fraction was carried out at100 000×g for 60 min at 4° C., and the membrane fractions wereresuspended in 100 μl PBS and stored at −70° C.

b. western blotting of membrane fractions.

4-20% Novex gradients were run under non-reducing, denaturing conditionsat 125V for 1.5 hr, and blotted in at 25V for 1.5 hr. Blotting wascarried out in the Novex apparatus exactly as recommended by themanufacturers using HYBOND C EXTRA® (membranes for binding protiens,Amersham).

c. Probing western blots.

Membranes were blocked for 45 min in MPBS and probed with 50 μg purifiedscFv in 5 ml MPBS for 1 hr at room temperature. Blots were washed inthree changes of PBST, followed by three changes of PBS. 9E10 at a 1:100dilution in MPBS was then incubated on the membrane for 1 hr at roomtemperature. Washing was carried out as before, and anti-mouse-IgG-HRPantibody then added at a dilution of 1:5000 in MPBS. Blots weredeveloped using ECL substrate (Amersham) and exposed to autoradiographicfilm.

Clone CD4E1 gave a band of approximately 29 kDa

Clone CD4D2 gave a band of approximately 31 kDa

Clone CD4A2 failed to give a specific band under denaturing gelconditions.

d. Immunocytochernistry (ICC) using scFv's on CD4+ cells.

Approximately 1×10⁵ CD4+ cells were spun onto poly-L-lysine subbedslides using a Cytospin (Serotech). Slides were blocked in MPBS for 2 hrat room temperature and a 1:10 dilution of the scFv in MPBS thenincubated on the slides for 1 hr at room temperature. Slides were washedin PBS and detection was achieved using 1:100 dilution of 9E10, followedby a 1:500 dilution of anti-mouse-HRP, both diluted in MPBS andincubated for 1 hr at room temperature, with washing in PBS betweenincubations. CD4E1 and CD4D2 both gave clear staining of the cellmembranes.

EXAMPLE 16 Demonstration of the use of Signal Transfer Selection toIdentify Novel Protien-Protien Interactions

To definitively identify the antigens which clones CD4A2, CD4E1 andCD4D2 recognise, a lambda gt11 cDNA expression library was constructedfrom mRNA from purified CD4+ cells and screened with purified ScFvs.

a. Isolation of messenger RNA

Messenger RNA was purified from a population of CD4 purified cells usinga QUICKPREP® Micro mRNA purification kit (Pharmacia). The mRNA waspurified following manufacturer's instructions. The method involvedlysis of the cells in a buffered aqueous solution containing guanidiniumthiocyanate and N-lauroyl sarcosine, the extract was then diluted threefold with an elution buffer which reduces the guanadinium concentrationto a level which is low enough to allow efficient hydrogen bondingbetween poly(A) tracts on the mRNA and the oligo(dT) attached tocellulose, but high enough to maintain complete inhibition of RNAses.The dilution step causes a number of proteins to precipitate, giving aninitial purification. The extract was clarified by short centrifugationat top speed in a minifuge and the supernatant transferred to amicrocentrifuge tube containing Oligo(dT)-cellulose. After 10 min,during which time the poly (A)+RNA binds to the oligo (dT)-cellulose,the tube was centrifuged at high speed for 10 sec, and the supernatantwas aspirated off the pelleted oligo (dT)-cellulose. Pelleted materialwas washed sequentially with 1 ml aliquots of high salt buffer and lowsalt buffer, each wash being accomplished by a process of resuspensionand brief centrifugation. After the last wash the pelleted material wasresuspended in 50 μl of low salt buffer and transferred to a MicroSpincolumn placed in a microcentrifuge tube, and the column was washed threetimes with 0.5 ml of low salt buffer. Finally, the polyadenylatedmateriel was eluted with prewarmed elution buffer (10 mM Tris-HCl(pH7.5), 1 mM EDTA). The mRNA was precipitated by addition of a glycogencarrier, potassium acetate and ethanol. After precipitation the mRNA wasrecovered by centrifugation and resuspended in DEPC treated water.

b. cDNA Synthesis

cDNA was synthesised from the CD4 mRNA using a cDNA synthesis kitsupplied by Amersham International. The detailed protocol booklet wasfollowed. The 1st strand synthesis reaction contained hexamer primersand reverse transcriptase with mRNA as template. Second strand synthesiswas carried out with Ribonuclease H and DNA polymerase, and aftersynthesis the ends of the cDNA were made blunt by treatment with T4 DNApolymerase. The cDNA was then purified by phenol/chloroform extraction.

c. Construction of cDNA library

cDNA was cloned into the lambda gt11 expression vector using Amersham'scDNA rapid adaptor ligation module (RPN 1712) and the cDNA rapid cloningmodule—gt11 (RPN1714) Adaptors were added to the cDNA to give EcoR1restriction cohesive ends, and cDNA with adaptors were separated fromfree adaptors by a column step. The adapted cDNA was then ligated intolambda gt11 vector then packaged using an in vitro packaging kit.Resultant reactions were titred to access the library size, which wasfound to be 7×10⁵.

d. Screening the cDNA expression library with scFv

For immunoscreening host cells (Y1090) were infected with phage from thelibrary and plated out on L top agarose. After 3.5 hours growth at 42°C., the plates were overlaid with nitrocellulose filters saturated with10 mM IPTG, an inducer of Lac Z gene expression, and incubated for afurther 3.5 hours at 37° C. During this time, the plaques aretransferred to the filter along with the β-galactosidase fusionproteins, released from the lytically infected cells. The filters werecarefully removed and washed briefly in PBS and then blocked in MPBST.Detection of positives was by sequential incubations with scfv ofinterest at a concentration of 10 μg/ml in MPBS, followed by 9E10 (1:100in MPBS) and then an anti-mouse HRP congugate (1:1000 in MPBS). Thefilters were washed between incubations in 3 changes of PBST. Signal wasdetected using an Enhanced Chemiluminescent system (Amersham ECL Kit).Plaques which were found to be positive from the first round ofscreening were picked and re-infected into a fresh culture of Y1090, andthe screening process repeated. This was carried out to ensure thereproducibility of the positive signal and to obtain clonal plaques.

e. Sequencing inserts from positive plaques.

Single plaques were picked into 100 μl SM buffer and left at 4° C.overnight. 5 μl of the eluted plaques was then taken and used astemplate for a standard 50 μl PCR reaction (0.5 μl TAQ Polymerase, 4 μl10 mM dNTP, 5 μl 10×PCR buffer, 2.5 μl of each primer (10 μM), made upto 50 μl with water). Primers used for sequencing were:

gt11screen5 (SEQ ID NO:35) 5′GAC TCC TGG AGC CCG gt11screen3 (SEQ IDNO:36) 3′GGT AGC GAC CGG CGC

PCR products were then cleaned up and used in sequencing reactions asdescribed previously (Example 2 part e), except that gt11screen5, andgt11screen3 were used as sequencing primers. Resultant nucleotidesequences were then aligned to the NCBI data base using the BLASTprogramme (Altschul et al., J. Mol. Biol. (1990) 215, 403-410.).

f. Results of the sequence alignments

lambda gtll Degree of scFv clone clone Homology identity CD4E1  2.1.1Rat CL-6  80% CD4A2  3.1.1 TRIP-4 (human) 100% CD4D2 10.1.1 26Sproteosome p31 (human) 100% CD4E1

This was found to recognise a lambda clone containing an insert whichhad homology to a rat protein called CL-6, which is an insulin-inducedgrowth response protein. This protein is a protein tyrosine phosphatase(PTP). PTP's are a family of intracellular and integral membranephosphatases which dephosphorylate tyrosine residues in proteins. PTP'shave been implicated in the control of normal and neoplastic growth andproliferation. PTPs have also been implicated in T-cell signaltransduction pathways, where they are involved in coupling receptors tothe generation of second messenger inositol triphosphate. The DNAfragment isolated here has 80% identity at the nucleotide level with therat CL-6 protein, and hence is probably the human homologue. CL-6 is anapproximately 30 kDa protein.

The rat gene CL-6 was identified by R. H. Diamond et al. (1993, Journalof Biological Chemistry 268, 15185-15192) as a gene which was induced inregenerating liver and insulin-treated Reuber H35 cells, a rat hepatomacell line which grows in response to physiological concentration ofinsulin and retains some properties of regenerating liver. CL-6 was oneof a panel of 41 novel growth response genes identified in this study,and was found to be the most abundant insulin-induced gene. CL-6 isinduced as an immediate-early gene in the liver cells, and itsimmediate-early induction during liver regeneration suggests that it isregulated by early stimuli, and not by insulin alone. CL-5 mRNAexpression was found to be highest in liver and kidney, but showed someexpression in most tissues. The CL-6 protein is predicted to be highlyhydrophobic, and may be a membrane-associated protein. CL-6 is likely tohave a role in the tissue-specific aspects of cellular growth, involvedin the maintenance of normal liver architecture or metabolism duringregeneration and foetal development.

CD4A2

This clone was found to recognise thyroid receptor interacting protein 4(TRIP4). Thyroid hormone receptors (TRs) are hormone-dependenttranscription factors that regulate expression of a variety of specifictarget genes. Thyroid interacting proteins are thought to play a role inmediating the TR's response to hormone binding.

CD4D2

This clone was found to recognise the p31 (31 kDa) subunit of the human26S proteosome. Proteosomes are involved in the ubiquitin-dependentproteolytic pathway and in antigen processing, and there is evidencethat they are found in close proxmity to, or associated with the plasmamembranes in vivo.

g. Summary of results

It has been demonstrated that the signal transfer selection procedurecan be used to select for antibodies, or other binding species, whichbind to antigens found in the vicinity of the original target antigen,but which do not recognise the target antigen itself. CD4A2, CD4E1 andCD4D2 are three examples of this. The antigens which these threeantibodies recognise have been identified by screening a cDNA expressionlibrary. The antigens identified by cDNA screening fit with thepredicted sizes of the antigens which CD4 E1 and CD4D2 bind to onwestern blotting i.e. the human homology of CL-6 (30 kDa), the p31subunit of the 26S proteosome (31 kDa). CDA2 recognises the TRIP4protein, which has an estimated molecular weight of 32 kDa. Theantibodies stain CD4+ cell membranes by ICC, as does MIP-1α, the ligandfor the CC-CKR5 receptor which was used to guide the signal transferseletion. Hence signal transfer selection has been used to identify apanel of antigens which are found in close proximity (probably up to 25nm) to MIP-1α receptors on the surface of CD4+ cells. This is ademonstration of the use of signal transfer selection as a means ofidentifying novel protein-protein interactions, and to identify novelgenes. The CL-6 gene has previously only been identified in rat, andsignal transfer selection has enabled the cloning of the human homologueof CL-6.

EXAMPLE 17 Biotinylation of CD4E1 Phage on the Cell Surface using MIP-1αto Direct the Biotinylation

Clone CD4E1 has been selected by virtue of the fact that it binds to anantigen found close to MIP-1α binding sites on CD4+ cell surfaces. Itshould therefore be possible to use biotinylated MIP-1α bound tostreptavidin-HRP to catalyse biotin tyramine deposition onto CD4E1 phagebound to the CD4+ cell surface to demonstrate that the CD4E1 antigen isnormally found in close association with MIP-1α receptors. This wastested by incubating cells with biotinylated MIP-1α, streptavidin-HRPand CD4E1 phage, treating with biotin tyramine and then recovering thebiotinylated phage and titring. Recovery of phage using this system wascompared to recovery when phage which bind at a site on the CD4+ cellsurface which is remote from the MIP-1α binding sites were incubatedwith the cells, or when a biotinylated ligand (biotinylated VCAM) whichbinds at another remote site on the CD4+ cell surface was used inconjunction with CD4E1 phage.

a. Biotinylation of phage on the cell surface.

CD4+ cells were purified as described in Example 9. CD4E1 phage, orphage from a CD4+ binding clone (CLA4) were prepared as described inExample 12. 1×10⁶ cells were incubated for 1 hr with 5 ng ofbiotinylated MIP-1α, or 5 ng of biotinylated VCAM in a total volume of100 μl PBS/BSA. Streptavidin-HRP (1:1000 dilution in PBS/BSA) was thenadded to the cells and incubated for 30 min. Cells were washed in PBS,and then 10¹¹ phage added in PBS/BSA and allowed to bind to the cellsfor 1 hr at room temperature. Cells were washed in PBS and then treatedwith biotin tyramine as described before. Cells were washed in PBS andthen lysed in PBS containing 0.1% Tween and biotinylated phage werecaptured on streptavidin-coated. Beads were washed three times in PBSTand three times in PBS, then infected directly into an exponentiallygrowing culture of E coli TG1.

b. Results of phase biotinylation.

The number of phage captured on beads was calculated from the titres. Ifno biotinylation reaction was carried out approximately 10⁷ CD4E1 andCLA4 phage were found to bind to the cell surface.

Phage Ligand Total Number of phage recovered CD4E1 MIP-1α 2000 CD4E1VCAM 800 CD4E1 — 400 CLA4 MIP-1α 600 CLA4 VCAM 800 CLA4 — 200

The number of phage recovered was at least 2.5 times higher when CD4E1phage was incubated with the MIP-1α, than the recovery attained in thevarious control samples. This provides indication that HBP-conjugatedMIP-1α is able to specifically biotinylate CD4E1 phage because CD4E1recognises an antigen which is found in close proximity (within 25 nm)to the MIP-1α receptor.

EXAMPLE 18 Use of Biotin Tyramine as a Signal Amplification Reagent inFlow Cytometry

Signal transfer can also be used as an amplification system forenhancing fluoresence signals in flow cytometry. This is achieved byallowing a HRP-conjugated antibody, or ligand to bind to cells. Cellscan then be treated with hydrogen peroxide and biotin tyramine, asdescribed for the selection procedure. This will cause biotin tyraminedeposition around the antibody, or ligand binding site on the cellsurface. Streptavidin-fluorescein (FITC) can then be added to the cells.This will bind to the newly deposited biotin on the cell surface andgive an enhancement in signal as compared to a standard FITC celllabelling protocol using FITC-conjugated antibody or ligand. This hasbeen shown to be the case by comparing the labelling achieved onpurified CD4+ lymphocytes using either an anti-CD4+ antibody, followedby anti-mouse-FITC, or by using anti-mouse-HRP followed by biotintyramine treatment and then streptavidin-FITC.

a. Cell labelling.

CD4+ lymphocytes were purified as described in Example 9. Cells wereincubated with the anti-CD4+ antibody (sigma), at a dilution of 1:1000in PBS/BSA. Cells were washed in PBS, and then either of the two secondantibodies (anti-mouse-FITC, or anti-mouse-HRP) were added to the cells,at a dilution of 1:1000 in PBS/BSA. 1×10⁵ cells were used per sample.Cells were washed in PBS and either detected directly (anti-mouse-FITC),or treated with biotin tyramine as described previously. Biotin tyraminewas added over a range of concentrations from 0.25 μg/ml up to 100μg/ml, in order co determine the concentration at which the optimalsignal enhancement occurred. After biotin tyramine treatment cells wereagain washed in PBS, then streptavidin-FITC was added at a diltuion of1:1000 in PBS. Cells were analysed by flow cytometry.

b. Flow Cytometry Results

The peak position (i.e. a measure of the fluorscence achieved) obtainedusing the different biotin tyramine concentrations was plotted. Theresults are shown in FIG. 4. As can be seen from the figure, optimalenhacement with biotin tyramine was obtaining using a concentration of12.5 μg/ml. The optimized peak position obtained using theanti-mouse-FITC second antibody was 60 fluorescence units, hence the useof biotin tyramine has efficiently enhanced this signal over 5-fold(from 60 to 330 fluorescence units).

EXAMPLE 19 Iteration of Biotin Tyramine Treatment to Give Further SignalEnhancement

Repeated rounds of biotin tyramine treatment may be carried out before afinal detection step, using streptavidin-FITC. The repeated rounds areachieved by an initial biotin tyramine treatment, followed by theaddition of streptavidin-HRP and then a further biotin tyraminetreatment. This example demonstrates the effective use of two rounds ofbiotin tyramine treatment to generate further signal enhancements. Amixed FICOLL® (a nonionic synthetic polymer of sucrose) purified cellpreparation (containing monocytes, lymphocytes and granulocytes) andlabelling with anti-CD36 antibody, which is a marker of monocytes, wasused here as a model system.

a. Cell labelling.

FICOLL® (a nonionic synthetic polymer of sucrose) purified cells (1×10⁶cells per sample) were incubated with anti-CD36 antibody for 30 min atroom temperature, diluted (1:1000) in PBS/BSA. Cells were washed inPBS/BSA and then incubated with an anti-mouse-HRP conjugate (1:1000 inPBS/BSA) for 30 min at room temperature. Cells were washed as before,and then treated with biotin tyramine at 12.5 μg/ml. Samples which wereto receive just one biotin tyramine treatment were then washed andincubated with streptavidin-FITC (1:1000 in PBS/BSA). The samples whichreceived a further treatment of biotin tyramine were incubated withstreptavidin-HRP (1:1000 in PBS/BSA) for 30 min at room temperature,then washed and treated with biotin tyramine as before. Cells werewashed again, and then incubated with streptavidin-FITC as before.

b. Results

Samples were analysed by flow cytometry and the fluorescence shiftsoverlayed, as shown in FIG. 5. As can be seen from the figure iterationof the biotin tyramine treatment results in a 2.5 fold shift in theaverage fluorescence level of the cells. This is to expected given theobservation that biotinylation may occurs over a range of up to 25 nmfrom the original site of HRP localisation. Assuming the first biotintyramine treatment biotinylates proteins in a circle of radius 25 nmfrom the HRP, then this would give an area of biotinylation of π25² nm²,which is 1963 nm². In the case of the second treatment with biotintyramine the biotin desposited in this area is then saturated withstreptavidin-HRP, so blocking binding of any streptavidin-FITC. Thebiotin tyramine treatment is repeated giving a further area ofbiotinylation of π50²−π25², which is 5887 nm², which is an area threetimes the size of the original circle of biotin deposition. This fitswith the experimentally observed observed fluorscence shift of around2.5 fold.

The fluorescence shift observed after iterations of biotin tyraminetreatment may be used to assess cellular copy numbers of cell surfaceproteins. If a protein is rare on a cell surface then the fluorescencesignal should carry on increasing with successive rounds of biotintyramine treatment until the cell surface is saturated. If a protein isexpressed at high copy number on a cell surface the fluorescence signalwill saturate sooner because the circles of biotinylation will overlap.

EXAMPLE 20 Use of Biotin Tyramine to Specifically BiotinylateSubpopulations of Cells to Allow Their Subsequent Purification

This example demonstrates using biotin tyramine to specificallybiotinylate subpopulations of cells within a complex mix and then tocapture the biotinylated cells to give an enriched population. Thesystem chosen here uses an anti-CD36 mouse monoclonal antibody(Immunotech) which is a monocyte cell surface marker. A mixture ofmonocytes, lymphocytes and granulocytes was purified from blood on aFICOLL® (a nonionic synthetic polymer of sucrose) density gradient.Lymphocytes and granulocytes do not express CD36, hence the antibodyshould specifically biotinylate monocytes. The technique is equallyapplicable to any molecule which binds cell surfaces, and to any celltype, virus particle, bead or other population of particles displayingan sbp member or epitope.

a. Purification of cells from buffy coat.

Adult buffy coat blood from Cambridge Blood Transfusion Service wasdiluted 1:2 with Dulbeccos PBS (Tissue culture grade) then loaded onto1077 density FICOLL HYPAQUE® (a nonionic synthetic polymer of sucrose,Sigma). This was then spun at 1500 rpm for 30 min at room temperaturewith brake off. Cells at the interface were removed and washed once withPBS. Red cells were removed by using a whole blood erythrocyte lysingkit from R&D systems (Cat. no. WL1000). Cells were resuspended in 5 mlof lysing reagent and left for 5 min at room temperature then spun at1000 rpm for 5 min and washed in 10 ml of wash reagent and again spun at1000 rpm for 5 min. Cells were resuspended in PBS/0.5% BSA/2mM EDTA(PBE) and then counted. In each of the following experiments 2.4×10⁶cells were used.

b. Antibody incubations and biotin tyramine treatment

Cells (2.4×10⁶) were incubated with mouse IgG₁ anti human CD36 antibody(Immunotech 0765) (2 μg/10⁵ cells) for 30 minutes at 4-8° C., washed inPBE and spun at 1000 rpm for 5 min. Incubation with goat anti-mouse HRPconjugated antibody (1:1000 dilution) was the same as for the anti CD36antibody. All antibodies were diluted in PBE. Cell pellets wereresuspended in 100 μl of 50 mM Tris-HCl pH7.4 with 2 μl biotin-tyramine(5 μg) and 1 μg of H₂O₂. This was left at room temperature for 10minutes and then washed with 5 ml of PBE.

c. Capture of biotinylated cells

This was carried out using streptavidin MACS® (magnetic cell sortingbeads, Miltenyi Biotec) as per manufacturer's instructions. Cells fromthe previous treatment was resuspended in 80 μl PBE with 20 μl of MACS®streptavidin (magnetic cell sorting beads, Cat, No. 481-01 MiltenyiBiotec). Incubation was for 15 min at 4° C. Cells were washed in PBE,resuspended in 100 μl of PBE and loaded onto a MACS® (magnetic cellsorting beads, Miltenyi Biotec) column enclosed in a MACS® (magneticcell sorting beads, Miltenyi Biotec) magnet. Cells were allowed to runin to the column, and then the column was washed with 2×1 ml of PBE toremove unbound cells. Cells were eluted from the column by removing thecolumn from the magnet, adding 1 ml of PBE, and then pushing the plungerinto the reservoir to push the PBE through the column. Cells were elutedinto an eppendorf and then spun at 4000 rpm for 5 min in amicrocentrifuge. Cell pellets were resuspended in 80 μl of PBE and 20 μlof anti-CD36 antibody conjugated to fluorescein (Immnunotech 0766).Cells were incubated in the dark at 4° C. for 20 minutes. Samples werethen analysed by flow cytometry.

d. Results

Anti-CD36 antibody, followed by anti-mouse-HRP and biotin tyraminetreatment was successful in biotinylating a subpopulation of cells whichwere subsequently captured on streptavidin beads. The captured cellswere found to be CD36 positive and were at the appropriate position byforward and side scatter in the flow analysis to be monocytes (FIG. 9).

EXAMPLE 21 Biotinylation of Phage Particles in Solution to ValidateBiotin-Tyramine Preparations

A phage preparation was made as described in Example 12 part a). Phageparticles were diluted to a titre of 1×10⁹ phage in 1 ml and 1 μl of aHRP-conjugated mouse Mab recognising the gene 8 protein HRP conjugate(Pharmacia) was added to the phage in solution. This was incubated atroom temperature for 1 hr, and the phage were then treated with biotintyramine, as described in Example 12 part b). Additional dilutions ofbiotin tyramine ranging from a 1:1000 dilution of the normal stocksolution, up to 100 fold excess over the normal concentrations remainedconstant. Biotinylated phage were then captured on 30 μl preblockedstreptavidin-coated beads and the beads washed as described before.Phage captured on beads were titred and the optimal biotin tyramineconcentration which gave maximal biotinylation was established. Theresults are shown in FIG. 7.

This provides a means of validating preparations of biotin tyramine, andallows comparison between different batches. The optimal biotin tyraminewas evaluated for two different preparations of biotin tyramine, and wasfound to be comparable.

TABLE 1 Expt 2nd Mab (HRP) No. phage in No. phage recovered % Eluate No.Phage type 1st Mab (1/2500) BT TEA eluate (× 10⁵) on beads (× 10³)recovered (i) CEA6 1/100 + + 5.6 4400 0.80 (ii) CEA6 1/1000 + + 3.9 10000.25 (iii) CEA6 1/10000 + + 9.1 1500 0.16 (iv) CEA6 1/100 + − 5.3 2800.05 (v) CEA6 1/100 − + 4.2 840 0.20 (vi) CEA6 — + + 4.6 440 0.10 (vii)OP1 1/100 + + 1.8 80 0.04

TABLE 2 Selec- Round tion 1 No. phage round phage No. phage in recovered% Eluate No. taken 1st Mab eluate (×10⁵) on beads (×10²) recovered 1A1/100 7.7 >300 >4 1B — 3.4 >200 >6 2 1A 1/100 1.8 4.13 0.23 2 1A 1/10001.4 3.64 0.26 2 1B — 2.8 1.11 0.04 2 1A — 1.5 1.87 0.12

TABLE 3 Round 1 No. Clones Selection phage taken screened CEA + ve %CEA + ve 1A — 94 3 4 1B — 94 0 0 2A 1A 48 13 27 2B 1A 65 11 17 2C 1A 484 8 2D 1B 25 1 4 1A = Selection with 1:100 dilution of anti-CEA mouseMab 1E = Selection with no anti-CEA Mab present 2A = Selection 1A takenand subjected to a second round of selection in the presence of a 1:100dilution of the anti-CEA mouse Mab 2B = Selection 1A taken and subjectedto a second round of selection in the presence of a 1:1000 dilution ofthe anti-CEA mouse Mab 2C = Selection 1A taken and subjected to a secondround of selection in the absence of the anti-CEA mouse Mab 2D =Selection 1B taken and subjected to a second round of selection in theabsense of the anti-CEA mouse Mab

TABLE 4 Clone Ic_(off) (s⁻¹) SS1A4 8.9 × 10⁻² SS1A11 7.2 × 10⁻² SS1G123.3 × 10⁻² SS22A8 7.8 × 10⁻² SS22B7 1.9 × 10⁻² SS22B1 1.3 × 10⁻² SS22D123.4 × 10⁻² SS22E4 7.5 × 10⁻² SS21B7 2.0 × 10⁻² SSDS1 3.0 × 10⁻² SS22A4ND SS21B7 ND

TABLE 5 Round 1 No. Clones Selection phage taken screened Mab + ve %Mab + ve 1A — 94 2 2 1B — 94 0 0 2A 1A 48 6 13 2B 1A 65 5 8 2C 1A 48 2 42D 1B 25 0 0 1A = Selection with 1:100 dilution of anti-CEA mouse Mab 1E= Selection with no anti-CEA Mab present 2A = Selection 1A taken andsubjected to a second round of selection in the presence of a 1:100dilution of the anti-CEA mouse Mab 2B = Selection 1A taken and subjectedto a second round of selection in the presence of a 1:1000 dilution ofthe anti-CEA mouse Mab 2C = Selection 1A taken and subjected to a secondround of selection in the absence of the anti-CEA mouse Mab 2D =Selection 1B taken and subjected to a second round of selection in theabsense of the anti-CEA mouse Mab

36 645 base pairs nucleic acid double linear 1 GAATTCCGGA AAAAACAAAATTCCTGTAAA ACAAATTAAC TCCAGGAACT TAAAATTTAC 60 TCCAAGACAT TTCCCTCAAAACAAAGCAAA AAACCCCAGC AAAGATCGTT ACATCACAAA 120 ACCAAACACA AAGACCAGCGGTCACAGGCA AGTTCCTCTA AGCTTCCATT CTGCTGACTG 180 GTGGCTTCCA TTTAAAAGGAGTCTTTTAAT CAAGCCACTT TCACAGAATT TAAAACAAAC 240 CAAACACATG TAAATTGCAAAATACAAAAA GGTAAATTTA TAAGTAAAAA TGACCAAACC 300 CACAAAACTG GAGTATTTCGAAGGTTGAGG GTTCAGTGGA GGGTGTAACA CGAAAGGAAC 360 TTCACAACTG AAAGAAATCATTGCCGAGTT TCCTCCAGGC AGCACTGAAA TGAATGGAGA 420 ACCTTCTCTC GAACATCTCACACGTTAAAA AAAATAAATA TTTAAGAGAT ACAAGGCTCA 480 GATTGGTTTT CATATACATTGCACTTGAAG TTTAAGACCC AATACTTGCA AATTAGGTCT 540 GGTATGGTTT ATGCCATTAAATGAATACAT TGTGCTCACC AATATCATTG ACTAGAAACA 600 CCACACGTTT AATGCAGTGCCATATGCAAT CTGTGACCGG AATTC 645 12 amino acids amino acid linear 2 GluPhe Arg Lys Lys Gln Asn Ser Cys Lys Thr Asn 1 5 10 45 amino acids aminoacid linear 3 Leu Gln Glu Leu Lys Ile Tyr Ser Lys Thr Phe Pro Ser LysGln Ser 1 5 10 15 Lys Lys Pro Gln Gln Arg Ser Leu His His Lys Thr LysHis Lys Asp 20 25 30 Gln Arg Ser Gln Ala Ser Ser Ser Lys Leu Pro Phe Cys35 40 45 5 amino acids amino acid linear 4 Leu Val Ala Ser Ile 1 5 50amino acids amino acid linear 5 Lys Glu Ser Phe Asn Gln Ala Thr Phe ThrGlu Phe Lys Thr Asn Gln 1 5 10 15 Thr His Val Asn Cys Lys Ile Gln LysGly Lys Phe Ile Ser Lys Asn 20 25 30 Asp Gln Thr His Lys Thr Gly Val PheArg Arg Leu Arg Val Gln Trp 35 40 45 Arg Val 50 19 amino acids aminoacid linear 6 His Glu Arg Asn Phe Thr Thr Glu Arg Asn His Cys Arg ValSer Ser 1 5 10 15 Arg Gln His 18 amino acids amino acid linear 7 Asn GluTrp Arg Thr Phe Ser Arg Thr Ser His Thr Leu Lys Lys Ile 1 5 10 15 AsnIle 32 amino acids amino acid linear 8 Glu Ile Gln Gly Ser Asp Trp PheSer Tyr Thr Leu His Leu Lys Phe 1 5 10 15 Lys Thr Gln Tyr Leu Gln IleArg Ser Gly Met Val Tyr Ala Ile Lys 20 25 30 8 amino acids amino acidlinear 9 Ile His Cys Ala His Gln Tyr His 1 5 6 amino acids amino acidlinear 10 Leu Glu Thr Pro His Val 1 5 7 amino acids amino acid linear 11Cys Ser Ala Ile Cys Asn Leu 1 5 83 amino acids amino acid linear 12 AsnSer Gly Lys Asn Lys Ile Pro Val Lys Gln Ile Asn Ser Arg Asn 1 5 10 15Leu Lys Phe Thr Pro Arg His Phe Pro Gln Asn Lys Ala Lys Asn Pro 20 25 30Ser Lys Asp Arg Tyr Ile Thr Lys Pro Asn Thr Lys Thr Ser Gly His 35 40 45Arg Gln Val Pro Leu Ser Phe His Ser Ala Asp Trp Trp Leu Pro Phe 50 55 60Lys Arg Ser Leu Leu Ile Lys Pro Leu Ser Gln Asn Leu Lys Gln Thr 65 70 7580 Lys His Met 9 amino acids amino acid linear 13 Ile Ala Lys Tyr LysLys Val Asn Leu 1 5 14 amino acids amino acid linear 14 Val Lys Met ThrLys Pro Thr Lys Leu Glu Tyr Phe Glu Gly 1 5 10 39 amino acids amino acidlinear 15 Gly Phe Ser Gly Gly Cys Asn Thr Lys Gly Thr Ser Gln Leu LysGlu 1 5 10 15 Ile Ile Ala Glu Phe Pro Pro Gly Ser Thr Glu Met Asn GlyGlu Pro 20 25 30 Ser Leu Glu His Leu Thr Arg 35 16 amino acids aminoacid linear 16 Ile Phe Lys Arg Tyr Lys Ala Gln Ile Gly Phe His Ile HisCys Thr 1 5 10 15 28 amino acids amino acid linear 17 Ser Leu Arg ProAsn Thr Cys Lys Leu Gly Leu Val Trp Phe Met Pro 1 5 10 15 Leu Asn GluTyr Ile Val Leu Thr Asn Ile Ile Asp 20 25 16 amino acids amino acidlinear 18 Lys His His Thr Phe Asn Ala Val Pro Tyr Ala Ile Cys Asp ArgAsn 1 5 10 15 8 amino acids amino acid linear 19 Ile Pro Glu Lys Thr LysPhe Leu 1 5 7 amino acids amino acid linear 20 Asn Lys Leu Thr Pro GlyThr 1 5 35 amino acids amino acid linear 21 Asn Leu Leu Gln Asp Ile SerLeu Lys Thr Lys Gln Lys Thr Pro Ala 1 5 10 15 Lys Ile Val Thr Ser GlnAsn Gln Thr Gln Arg Pro Ala Val Thr Gly 20 25 30 Lys Phe Leu 35 15 aminoacids amino acid linear 22 Ala Ser Ile Leu Leu Thr Gly Gly Phe His LeuLys Gly Val Phe 1 5 10 15 7 amino acids amino acid linear 23 Ser Ser HisPhe His Arg Ile 1 5 13 amino acids amino acid linear 24 Asn Lys Pro AsnThr Cys Lys Leu Gln Asn Thr Lys Arg 1 5 10 25 amino acids amino acidlinear 25 Pro Asn Pro Gln Asn Trp Ser Ile Ser Lys Val Glu Gly Ser ValGlu 1 5 10 15 Gly Val Thr Arg Lys Glu Leu His Asn 20 25 13 amino acidsamino acid linear 26 Lys Lys Ser Leu Pro Ser Phe Leu Gln Ala Ala Leu Lys1 5 10 33 amino acids amino acid linear 27 Met Glu Asn Leu Leu Ser AsnIle Ser His Val Lys Lys Asn Lys Tyr 1 5 10 15 Leu Arg Asp Thr Arg LeuArg Leu Val Phe Ile Tyr Ile Ala Leu Glu 20 25 30 Val 6 amino acids aminoacid linear 28 Asp Pro Ile Leu Ala Asn 1 5 7 amino acids amino acidlinear 29 Val Trp Tyr Gly Leu Cys His 1 5 28 amino acids amino acidlinear 30 Met Asn Thr Leu Cys Ser Pro Ile Ser Leu Thr Arg Asn Thr ThrArg 1 5 10 15 Leu Met Gln Cys His Met Gln Ser Val Thr Gly Ile 20 25 15amino acids amino acid linear 31 Gly Gly Gly Gly Ser Gly Gly Gly Gly SerGly Gly Gly Gly Ser 1 5 10 15 20 amino acids amino acid linear 32 MetAsp Tyr Gln Val Ser Ser Pro Ile Tyr Asp Ile Asn Tyr Tyr Thr 1 5 10 15Ser Glu Pro Cys 20 10 amino acids amino acid linear 33 Pro Met Pro HisAla Glu Gly Lys Ser Thr 1 5 10 14 amino acids amino acid linear 34 GlyAla Ala Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Met 1 5 10 15 base pairsnucleic acid single linear 35 GACTCCTGGA GCCCG 15 15 base pairs nucleicacid single linear 36 CGCGGCCAGC GATGG 15

We claim:
 1. A method of obtaining a peptide or polypeptide, the methodcomprising providing in a common medium: label molecules; a markerligand able to bind a member of a specific binding pair (sbp), whichspecific binding pair has a first sbp member and a second sbp member,and wherein said marker ligand is able to bind said second sbp member;said second sbp member; an enzyme able to catalyze binding of said labelmolecules to proteins, peptides or ligands, said enzyme being conjugatedto said marker ligand; causing or allowing binding of said marker ligandto said second sbp member; and causing or allowing binding of said labelmolecules to proteins, peptides or ligands in the vicinity of saidmarker ligand bound to said second sbp member thereby providing labeledproteins, peptides or ligands; wherein said proteins, peptides orligands to which said label molecules bind comprise a sbp member (“firstsbp member”) which binds said second sbp member or comprise a sbp member(“further sbp member”) which binds a protein, peptide or ligand in thevicinity of said second sbp member, and said first sbp member or saidfurther sbp member is displayed on the surface of a virus particle; themethod further comprising: isolating and/or purifying said particle fromthe medium following binding of said first sbp member to said second sbpmember or binding of said further sbp member to a protein, peptide orligand in the vicinity of said second sbp member, and labeling of saidparticle and/or said displayed first or further sbp member; obtainingnucleic acid from an isolated and/or purified particle; and producing byexpression from nucleic acid with the sequence of said nucleic acidobtained from said particle or a sequence modified by the addition,insertion, deletion and/or substitution of one or more nucleotides anencoded peptide or polypeptide.
 2. A method according to claim 1 whereinsaid particle is isolated and/or purified by means of a member of aspecific binding pair able to bind said labeled particle and/ordisplayed first or further sbp member.
 3. A method according to claim 1wherein the peptide or polypeptide produced by expression from saidnucleic acid is formulated into a composition including at least oneadditional component.
 4. A method according to claim 3 wherein thecomposition includes a pharmaceutically acceptable vehicle or carrier.